Production of hydroxylated fatty acids in genetically modified plants

ABSTRACT

This invention relates to plant fatty acyl hydroxylases. Methods to use conserved amino acid or nucleotide sequences to obtain plant fatty acyl hydroxylases are described. Also described is the use of cDNA clones encoding a plant hydroxylase to produce a family of hydroxylated fatty acids in transgenic plants. In addition, the use of genes encoding fatty acid hydroxylases or desaturases to alter the level of lipid fatty acid unsaturation in transgenic plants is described.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 08/530,862, filed Sep. 20, 1995, the entirecontents of which are hereby incorporated by reference and relied upon.

GOVERNMENT RIGHTS

[0002] The invention described herein was made in the course of workunder grant number DE-FG02-94ER20133 from the U.S. Department of Energyand grant No. MCB9305269 from the National Science Foundation.Therefore, the U.S. Government has certain rights under this invention.

TECHNICAL FIELD

[0003] The present invention concerns the identification of nucleic acidsequences and constructs, and methods related thereto, and the use ofthese sequences and constructs to produce genetically modified plantsfor the purpose of altering the fatty acid composition of plant oils,waxes and related compounds.

DEFINITIONS

[0004] The subject of this invention is a class of enzymes thatintroduce a hydroxyl group into several different fatty acids resultingin the production of several different kinds of hydroxylated fattyacids. In particular, these enzymes catalyze hydroxylation of oleic acidto 12-hydroxy oleic acid and icosenoic acid to 14-hydroxy icosenoicacid. Other fatty acids such as palmitoleic and erucic acids may also besubstrates. Since it is not possible to refer to the enzyme by referenceto a unique substrate or product, we refer to the enzyme throughout askappa hydroxylase to indicate that the enzyme introduces the hydroxylthree carbons distal (i.e., away from the carboxyl carbon of the acylchain) from a double bond located near the center of the acyl chain.

[0005] The following fatty acids are also the subject of this invention:ricinoleic acid, 12-hydroxyoctadec-cis-9-enoic acid (12OH-18:1^(cisΔ9));lesquerolic acid, 14-hydroxy-cis-11-icosenoic acid (14OH-20:1^(cisΔ11));densipolic acid, 12-hydroxyoctadec-cis-9,15-dienoic acid(12OH-18:2^(cisΔ9,15)); auricolic acid,14-hydroxy-cis-11,17-icosadienoic acid (14OH-20:2^(cisΔ11,17));hydroxyerucic, 16-hydroxydocos-cis-13-enoic acid (16OH-22:1^(cisΔ13));hydroxypalmitoleic, 12-hydroxyhexadec-cis-9-enoic (12OH-16:1^(cisΔ9));icosenoic acid (20:1^(cisΔ11)). It will be noted that icosenoic acid isspelled eicosenoic acid in some countries.

BACKGROUND

[0006] Extensive surveys of the fatty acid composition of seed oils fromdifferent species of higher plants have resulted in the identificationof at least 33 structurally distinct monchydroxylated plant fatty acids,and 12 different polyhydroxylated fatty acids that are accumulated byone or more plant species (reviewed by van de Loo et al. 1993).Ricinoleic acid, the principal constituent of the seed oil from thecastor plant Ricinus communis (L.), is of commercial importance. We havepreviously described the cloning of a gene from this species thatencodes a fatty acid hydroxylase, and the use of this gene to producericinoleic acid in transgenic plants of other species (see U.S. patentapplication Ser. No. 08/320,982, filed Oct. 11, 1994). The scientificevidence supporting the claims in that patent application weresubsequently published (van de Loo et al., 1995).

[0007] The use of the castor hydroxylase gene to also produce otherhydroxylated fatty acids such as lesquerolic acid, densipolic acid,hydroxypalmitoleic, hydroxyerucic and auricolic acid in transgenicplants is the subject of this invention. In addition, the identificationof a gene encoding a homologous hydroxylase from Lesquerella fendleri,and the use of this gene to produce these hydroxylated fatty acids intransgenic plants is the subject of this invention.

[0008] Castor is a minor oilseed crop. Approximately 50% of the seedweight is oil (triacylglycerol) in which 85-90% of total fatty acids arethe hydroxylated fatty acid, ricinoleic acid. Oil pressed or extractedfrom castor seeds has many industrial uses based upon the propertiesendowed by the hydroxylated fatty acid. The most important uses areproduction of paints and varnishes, nylon-type synthetic polymers,resins, lubricants, and cosmetics (Atsmon 1989).

[0009] In addition to oil, the castor seed contains the extremely toxicprotein ricin, allergenic proteins, and the alkaloid ricinine. Theseconstituents preclude the use of the untreated seed meal (following oilextraction) as a livestock feed, normally an important economic aspectof oilseed utilization. Furthermore, with the variable nature of castorplants and a lack of investment in breeding, castor has few favorableagronomic characteristics.

[0010] For a combination of these reasons, castor is no longer grown inthe United States and the development of an alternative domestic sourceof hydroxylated fatty acids would be attractive. The production ofricinoleic acid, the important constituent of castor oil, in anestablished oilseed crop through genetic engineering would be aparticularly effective means of creating a domestic source.

[0011] Because there is no practical source of lesquerolic, densipolicand auricolic acids from plants that are adapted to modern agriculturalpractices, there is currently no large-scale use of these fatty acids byindustry. However, the fatty acids would have uses similar to those ofricinoleic acid if they could be produced in large quantities atcomparable cost to other plant-derived fatty acids (Smith 1985).

[0012] Plant species, such as certain species in the genus Lesquerella,that accumulate a high proportion of these fatty acids, have not beendomesticated and are not currently considered a practical source offatty acids (Hirsinger, 1989). This invention represents a useful steptoward the eventual production of these and other hydroxylated fattyacids in transgenic plants of agricultural importance.

[0013] The taxonomic relationships between plants having similar oridentical kinds of unusual fatty acids have been examined (van de Loo etal., 1993). In some cases, particular fatty acids occur mostly or solelyin related taxa. In other cases there does not appear to be a directlink between taxonomic relationships and the occurrence of unusual fattyacids. In this respect, ricinoleic acid has now been identified in 12genera from 10 families (reviewed in van de Loo et al., 1993). Thus, itappears that the ability to synthesize hydroxylated fatty acids hasevolved several times independently during the radiation of theangiosperms. This suggested to us that the enzymes which introducehydroxyl groups into fatty acids arose by minor modifications of arelated enzyme.

[0014] Indeed, as shown herein, the sequence similarity between Δ12fatty acid desaturases and the kappa hydroxylase from castor is so highthat it is not possible to unambiguously determine whether a particularenzyme is a desaturase or a hydroxylase on the basis of evidence in thescientific literature. Similarly, a patent application (PCT/US93/09987)that purports to teach the isolation and use of Δ12 fatty aciddesaturases does not teach how to distinguish a hydroxylase from adesaturase. In view of the importance of being able to distinguishbetween these activities for the purpose of genetic engineering of plantoils, the utility of that application is limited to the severalinstances where direct experimental evidence (e.g., altered fatty acidcomposition in transgenic plants) was presented to support theassignment of function. A method for distinguishing between fatty aciddesaturases and fatty acid hydroxylases on the basis of amino acidsequence of the enzyme is also a subject of this invention.

[0015] A feature of hydroxylated or other unusual fatty acids is thatthey are generally confined to seed triacylglycerols, being largelyexcluded from the polar lipids by unknown mechanisms (Battey andOhlrogge 1989; Prasad et al., 1987). This is particularly intriguingsince diacylglycerol is a precursor of both triacylglycerol and polarlipid. With castor microsomes, there is some evidence that the pool ofricinoleoyl-containing polar lipid is minimized by a preference ofdiacylglycerol acyltransferase for ricinoleate-containingdiacylglycerols (Bafor et al. 1991). Analyses of vegetative tissues havegenerated few reports of unusual fatty acids, other than those occurringin the cuticle. The cuticle contains various hydroxylated fatty acidswhich are interesterified to produce a high molecular weight polyesterwhich serves a structural role. A small number of other exceptions existin which unusual fatty acids are found in tissues other than the seed.

[0016] The biosynthesis of ricinoleic acid from oleic acid in thedeveloping endosperm of castor (Ricinus communis) has been studied by avariety of methods. Morris (1967) established in double-labeling studiesthat hydroxylation occurs directly by hydroxyl substitution rather thanvia an unsaturated-, keto- or epoxy-intermediate. Hydroxylation usingoleoyl-CoA as precursor can be demonstrated in crude preparations ormicrosomes, but activity in microsomes is unstable and variable, andisolation of the microsomes involved a considerable, or sometimescomplete loss of activity (Galliard and Stumpf, 1966; Moreau and Stumpf,1981). Oleic acid can replace oleoyl-CoA as a precursor, but only in thepresence of CoA, MG²⁺ and ATP (Galliard and Stumpf, 1966) indicatingthat activation to the acyl-CoA is necessary. However, no radioactivitycould be detected in ricinoleoyl-CoA (Moreau and Stumpf, 1981). Theseand more recent observations (Bafor et al., 1991) have been interpretedas evidence that the substrate for the castor oleate hydroxylase isoleic acid esterified to phosphatidylcholine or another phospholipid.

[0017] The hydroxylase is sensitive to cyanide and azide, and dialysisagainst metal chelators reduces activity, which could be restored byaddition of FeSO₄, suggesting iron involvement in enzyme activity(Galliard and Stumpf, 1966). Ricinoleic acid synthesis requiresmolecular oxygen (Galliard and Stumpf, 1966; Moreau and Stumpf 1981) andrequires NAD(P)H to reduce cytochrome b5 which is thought to be theintermediate electron donor for the hydroxylase reaction (Smith et al.,1992). Carbon monoxide does not inhibit hydroxylation, indicating that acytochrome P450 is not involved (Galliard and Stumpf, 1966; Moreau andStumpf 1981). Data from a study of the substrate specificity of thehydroxylase show that all substrate parameters (i.e., chain length anddouble bond position with respect to both ends) are important;deviations in these parameters caused reduced activity relative to oleicacid (Howling et al., 1972). The position at which the hydroxyl wasintroduced, however, was determined by the position of the double bond,always being three carbons distal. Thus, the castor acyl hydroxylaseenzyme can produce a family of different hydroxylated fatty acidsdepending on the availability of substrates. Thus, as a matter ofconvenience, we refer to the enzyme throughout as a kappa hydroxylase(rather than an oleate hydroxylase) to indicate the broad substratespecificity.

[0018] The castor kappa hydroxylase has many superficial similarities tothe microsomal fatty acyl desaturases (Browse and Somerville, 1991). Inparticular, plants have a microsomal oleate desaturase active at the Δ12position. The substrate of this enzyme (Schmidt et al., 1993) and of thehydroxylase (Bafor et al., 1991) appears to be a fatty acid esterifiedto the sn-2 position of phosphatidylcholine. When oleate is thesubstrate, the modification occurs at the same position (Δ12) in thecarbon chain, and requires the same cofactors, namely electrons fromNADH via cytochrome b₅ and molecular oxygen. Neither enzyme is inhibitedby carbon monoxide (Moreau and Stumpf, 1981), the characteristicinhibitor of cytochrome P450 enzymes.

[0019] There do not appear to have been any published biochemicalstudies of the properties of the hydroxylase enzyme(s) in Lesquerella.

[0020] Conceptual Basis of the Invention

[0021] In U.S. patent application Ser. No. 08/320,982, we described theuse of a cDNA clone from castor for the production of ricinoleic acid intransgenic plants. As noted above, biochemical studies by others hadsuggested that the castor hydroxylase may not have strict specificityfor oleic acid but would also catalyze hydroxylation of other fattyacids such as icosenoic acid (20:1^(cisΔ11)) (Howling et al., 1972).Based on these studies, our previous application Ser. No. 08/320,982noted in Example 2 that the expression of the castor hydroxylase intransgenic plants of species such as Brassica napus and Arabidopsisthaliana that accumulate fatty acids such as icosenoic acid(20:1^(cisΔ11)) and erucic acid (13-docosenoic acid; 22:1^(cisΔ13))would be expected to accumulate some of the hydroxylated derivatives ofthese fatty acids due to the activity of the hydroxylase on these fattyacids. We have now obtained additional direct evidence for such a claimbased on the production of ricinoleic, lesquerolic, densipolic andauricolic fatty acids in transgenic Arabidopsis plants and have includedsuch evidence herein as Example 1.

[0022] In Example 3 of the previous application, we taught the variousmethods by which the castor hydroxylase clone and sequences derivedthereof could be used to identify other hydroxylase clones from plantspecies such as Lesquerella fendleri that are known to accumulatehydroxylated fatty acids in seed oils. In this continuation we haveprovided an example of the use of that aspect of the invention for theisolation of a novel hydroxylase gene from Lesquerella fendleri.

[0023] In view of the high degree of sequence similarity between Δ12fatty acid desaturases and the castor hydroxylase (van de Loo et al.,1995), the validity of claims (e.g., PCT WO 94/11516) for the use ofdesaturase or hydroxylase genes or sequences derived therefrom for theidentification of genes of identical function from other species must beviewed with skepticism. In this application, we teach a method by whichhydroxylase genes can be distinguished from desaturases and describemethods by which Δ12 desaturases can be converted to hydroxylases by themodification of the gene encoding the desaturases. A mechanistic basisfor the similar reaction mechanisms of desaturases and hydroxylases waspresented in the earlier patent application (Ser. No. 08/320,982).Briefly, the available evidence suggests that fatty acid desaturaseshave a similar reaction mechanism to the bacterial enzyme methanemonooxygenase which catalyses a reaction involving oxygen-atom transfer(CH₄→CH₃OH) (van de Loo et al., 1993). The cofactor in the hydroxylasecomponent of methane monooxygenase is termed a μ-oxo bridged diironcluster (FeOFe). The two iron atoms of the FeOFe cluster are liganded byprotein-derived nitrogen or oxygen atoms, and are tightly redox-coupledby the covalently-bridging oxygen atom. The FeOFe cluster accepts twoelectrons, reducing it to the diferrous state, before oxygen binding.Upon oxygen binding, it is likely that heterolytic cleavage also occurs,leading to a high valent oxoiron reactive species that is stabilized byresonance rearrangements possible within the tightly coupled FeOFecluster. The stabilized high-valent oxoiron state of methanemonooxygenase is capable of proton extraction from methane, followed byoxygen transfer, giving methanol. The FeOFe cofactor has been shown tobe directly relevant to plant fatty acid modifications by thedemonstration that castor stearoyl-ACP desaturase contains this type ofcofactor (Fox et al., 1993).

[0024] On the basis of the foregoing considerations, we hypothesizedthat the castor oleate hydroxylase is a structurally modified fatty acyldesaturase, based upon three arguments. The first argument involves thetaxonomic distribution of plants containing ricinoleic acid. Ricinoleicacid has been found in 12 genera of 10 families of higher plants(reviewed in van de Loo et al., 1993). Thus, plants in which ricinoleicacid occurs are found throughout the plant kingdom, yet close relativesof these plants do not contain the unusual fatty acid. This patternsuggests that the ability to synthesize ricinoleic acid has arisen (andbeen lost) several times independently, and is therefore a quite recentdivergence. In other words, the ability to synthesize ricinoleic acidhas evolved rapidly, suggesting that a relatively minor genetic changein the structure of the ancestral enzyme was necessary to accomplish it.

[0025] The second argument is that many biochemical properties of castorkappa hydroxylase are similar to those of the microsomal desaturases, asdiscussed above (e.g., both preferentially act on fatty acids esterifiedto the sn-2 position of phosphatidylcholine, both use cytochrome b5 asan intermediate electron donor, both are inhibited by cyanide, bothrequire molecular oxygen as a substrate, both are thought to be locatedin the endoplasmic reticulum).

[0026] The third argument stems from the discussion of oxygenasecofactors above, in which it is suggested that the plant membrane boundfatty acid desaturases may have a μ-oxo bridged diiron cluster-typecofactor, and that such cofactors are capable of catalyzing both fattyacid desaturations and hydroxylations, depending upon the electronic andstructural properties of the protein active site.

[0027] Taking these three arguments together, it was hypothesized thatkappa hydroxylase of castor endosperm is homologous to the microsomaloleate Δ12 desaturase found in all plants. The evidence supporting thishypothesis was disclosed in the previous patent application (Ser. No.08/320,982). A number of genes encoding microsomal Δ12 desaturases fromvarious species have recently been cloned (Okuley et al., 1994) andsubstantial information about the structure of these enzymes is nowknown (Shanklin et al. 1994). Hence, in the following invention we teachhow to use structural information about fatty acyl desaturases toisolate kappa hydroxylase genes of this invention. This example teachesthe method by which any carbon-monoxide insensitive plant fatty acylhydroxylase gene can be identified by one skilled in the art.

[0028] An unpredicted outcome of our studies on the castor hydroxylasegene in transgenic Arabidopsis plants was the discovery that expressionof the hydroxylase leads to increased accumulation of oleic acid in seedlipids. Because of the low nucleotide sequence homology between thecastor hydroxylase and the Δ12-desaturase (about 67%), we consider itunlikely that this effect is due to silencing (also calledsense-suppression or cosuppression) of the expression of the desaturasegene by the hydroxylase gene. Whatever the basis for the effect, thisinvention teaches the use of hydroxylase genes to alter the level offatty acid unsaturation in transgenic plants. On the basis of ahypothesis about the mechanisms of the effect, this invention alsoteaches the use of genetically modified hydroxylase and desaturase genesto achieve directed modification of fatty acid unsaturation levels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGS. 1A-D show the mass spectra of hydroxy fatty acids standards(FIG. 1A, O-TMS-methylricinoleate; FIG. 1B, O-TMS-methyl densipoleate;FIG. 1C, O-TMS-methyl-lesqueroleate; and FIG. 1D,O-TMS-methylauricoleate).

[0030]FIG. 2 shows the fragmentation pattern of trimethylsilylatedmethyl esters of hydroxy fatty acids.

[0031]FIG. 3A shows the gas chromatogram of fatty acids extracted fromseeds of wild type Arabidopsis plants. FIG. 3B shows the gaschromatogram of fatty acids extracted from seeds of transgenicArabidopsis plants containing the fah12 hydroxylase gene. The numbersindicate the following fatty acids: [1] 16:0; [2] 18:0; [3] 18:1cisΔ9;[4] 18:2^(cisΔ9,12); [5] 20:0; [6] 20:1^(cisΔ11); [7]18:3^(cisΔ9,12,15); [8] 22:1^(cisΔ13); [9] 24:1^(cisΔ13); [10]ricinoleic acid; [11] densipolic acid; [12] lesquerolic acid; [13]auricolic acid.

[0032] FIGS. 4A-D show the mass spectra of novel fatty acids found inseeds of transgenic plants. FIG. 4A shows the mass spectrum of peak 10from FIG. 3B. FIG. 4B shows the mass spectrum of peak 11 from FIG. 3B.FIG. 4C shows the mass spectrum of peak 12 from FIG. 3B. FIG. 4D showsthe mass spectrum of peak 13 from FIG. 3B.

[0033]FIG. 5 shows the nucleotide sequence of pLesq2 (SEQ ID NO:1).

[0034]FIG. 6 shows the nucleotide sequence of pLesq3 (SEQ ID NO:2).

[0035]FIG. 7 shows a Northern blot of total RNA from seeds of L.fendleri probed with pLesq2 or pLesq3. S. indicates RNA is from seeds;L, indicates RNA is from leaves.

[0036] FIGS. 8A-B show the nucleotide sequence of genomic clone encodingpLesq-HYD (SEQ ID NO:3), and the deduced amino acid sequence ofhydroxylase enzyme encoded by the gene (SEQ ID NO:4).

[0037] FIGS. 9A-B show multiple sequence alignment of deduced amino acidsequences for kappa hydroxylases and microsomal Δ12 desaturases.Abbreviations are: Rcfah12, fah12 hydroxylase gene from R. communis (vande Loo et al., 1995); Lffah12, kappa hydroxylase gene from L. fendleri;Atfad2, fad2 desaturase from Arabidopsis thaliana (Okuley et al., 1994);Gmfad2-1, fad2 desaturase from Glycine max (GenBank accession numberL43920); Gmfad2-2, fad2 desaturase from Glycine max (Genbank accessionnumber L43921); Zmfad2, fad2 desaturase from Zea mays (PCT/US93/09987);Rcfad2, fragment of fad2 desaturase from R. communis (PCT/US93/09987);Bnfad2, fad2 desaturase from Brassica napus (PCT/US93/09987);LFFAH12.AMI, SEQ ID NO:4; FAH12.AMI, SEQ ID NO:5; ATFAD2.AMI, SEQ IDNO:6; BNFAD2.AMI, SEQ ID NO:7; GMFAD2-1.AMI, SEQ ID NO:8; GMFAD2-2.AMI,SEQ ID NO:9; ZMFAD2.AMI, SEQ ID NO:10; and RCFAD2.AMI, SEQ ID NO:11.

[0038]FIG. 10 shows a Southern blot of genomic DNA from L. fendleriprobed with pLesq-HYD. E=EcoRI, H=HindIII, X=XbaI.

[0039]FIG. 11 shows a map of binary Ti plasmid pSLJ44024.

[0040]FIG. 12 shows a map of plasmid pYES2.0

[0041]FIG. 13 shows part of a gas chromatogram of derivatized fattyacids from yeast cells that contain plasmid pLesqYes in which expressionof the hydroxylase gene was induced by addition of galactose to thegrowth medium. The arrow points to a peak that is not present inuninduced cells. The lower part of the figure is the mass spectrum ofthe peak indicated by the arrow.

SUMMARY OF THE INVENTION

[0042] This invention relates to plant fatty acyl hydroxylases. Methodsto use conserved amino acid or nucleotide sequences to obtain plantfatty acyl hydroxylases are described. Also described is the use of cDNAclones encoding a plant hydroxylase to produce a family of hydroxylatedfatty acids in transgenic plants.

[0043] In a first embodiment, this invention is directed to recombinantDNA constructs which can provide for the transcription or transcriptionand translation (expression) of the plant kappa hydroxylase sequence. Inparticular, constructs which are capable of transcription ortranscription and translation in plant host cells are preferred. Suchconstructs may contain a variety of regulatory regions includingtranscriptional initiation regions obtained from genes preferentiallyexpressed in plant seed tissue. In a second aspect, this inventionrelates to the presence of such constructs in host cells, especiallyplant host cells which have an expressed plant kappa hydroxylasetherein.

[0044] In yet another aspect, this invention relates to a method forproducing a plant kappa hydroxylase in a host cell or progeny thereofvia the expression of a construct in the cell. Cells containing a plantkappa hydroxylase as a result of the production of the plant kappahydroxylase encoding sequence are also contemplated herein.

[0045] In another embodiment, this invention relates to methods of usinga DNA sequence encoding a plant kappa hydroxylase for the modificationof the proportion of hydroxylated fatty acids produced within a cell,especially plant cells. Plant cells having such a modified hydroxylatedfatty acid composition are also contemplated herein.

[0046] In a further aspect of this invention, plant kappa hydroxylaseproteins and sequences which are related thereto, including amino acidand nucleic acid sequences, are contemplated. Plant kappa hydroxylaseexemplified herein includes a Lesquerella fendleri fatty acidhydroxylase. This exemplified fatty acid hydroxylase may be used toobtain other plant fatty acid hydroxylases of this invention.

[0047] In a further aspect of this invention, a nucleic acid sequencewhich directs the seed specific expression of an associated polypeptidecoding sequence is described. The use of this nucleic acid sequence orfragments derived thereof, to obtain seed-specific expression in higherplants of any coding sequence is contemplated herein.

[0048] In a further aspect of this invention, the use of genes encodingfatty acyl hydroxylases of this invention are used to alter the amountof fatty acid unsaturation of seed lipids. We further envision the useof genetically modified hydroxylase and desaturase genes to achievedirected modification of fatty acid unsaturation levels.

DETAILED DESCRIPTION OF THE INVENTION

[0049] A genetically transformed plant of the present invention whichaccumulates hydroxylated fatty acids can be obtained by expressing thedouble-stranded DNA molecules described in this application.

[0050] A plant fatty acid hydroxylase of this invention includes anysequence of amino acids, such as a protein, polypeptide or peptidefragment, or nucleic acid sequences encoding such polypeptides,obtainable from a plant source which demonstrates the ability tocatalyze the production of ricinoleic, lesquerolic, hydroxyerucic(16-hydroxydocos-cis-13-enoic acid) or hydroxypalmitoleic(12-hydroxyhexadec-cis-9-enoic) from CoA, ACP or lipid-linked monoenoicfatty acid substrates under plant enzyme reactive conditions. By “enzymereactive conditions” is meant that any necessary conditions areavailable in an environment (i.e., such factors as temperature, pH, lackof inhibiting substances) which will permit the enzyme to function.

[0051] Preferential activity of a plant fatty acid hydroxylase toward aparticular fatty acyl substrate is determined upon comparison ofhydroxylated fatty acid product amounts obtained per different fattyacyl substrates. For example, by “oleate preferring” is meant that thehydroxylase activity of the enzyme preparation demonstrates a preferencefor oleate-containing substrates over other substrates. Although theprecise substrate of the castor fatty acid hydroxylase is not known, itis thought to be a monounsaturated fatty acid moiety which is esterifiedto a phospholipid such as phosphatidylcholine. However, it is alsopossible that monounsaturated fatty acids esterified tophosphatidylethanolamine, phosphatidic acid or a neutral lipid such asdiacylglycerol or a Coenzyme-A thicester may also be substrates.

[0052] As noted above, significant activity has been observed inradioactive labelling studies using fatty acyl substrates other thanoleate (Howling et al., 1972) indicating that the substrate specificityis for a family of related fatty acyl compounds. Because the castorhydroxylase introduces hydroxy groups three carbons from a double bond,proximal to the methyl carbon of the fatty acid, we term the enzyme akappa hydroxylase for convenience. Of particular interest, we envisionthat the castor kappa hydroxylase may be used for production of12-hydroxy-9-octadecenoic acid (ricinoleate), 12-hydroxy-9-hexadecenoicacid, 14-hydroxy-11-eicosenoic acid, 16-hydroxy-13-docosanoic acid,9-hydroxy-6-octadecenoic acid by expression in plants species whichproduce the non-hydroxylated precursors. We also envision production ofadditionally modified fatty acids such as12-hydroxy-9,15-octadecadienoic acid that result from desaturation ofhydroxylated fatty acids (e.g., 12-hydroxy-9-octadecenoic acid in thisexample).

[0053] We also envision that future advances in the genetic engineeringof plants will lead to production of substrate fatty acids, such asicosenoic acid esters, and palmitoleic acid esters in plants that do notnormally accumulate such fatty acids. We envision that the inventiondescribed herein may be used in conjunction with such futureimprovements to produce hydroxylated fatty acids of this invention inany plant species that is amenable to directed genetic modification.Thus, the applicability of this invention is not limited in ourconception only to those species that currently accumulate suitablesubstrates.

[0054] As noted above, a plant kappa hydroxylase of this invention willdisplay activity towards various fatty acyl substrates. Duringbiosynthesis of lipids in a plant cell, fatty acids are typicallycovalently bound to acyl carrier protein (ACP), coenzyme A (CoA) orvarious cellular lipids. Plant kappa hydroxylases which displaypreferential activity toward lipid-linked acyl substrate are especiallypreferred because they are likely to be closely associated with normalpathway of storage lipid synthesis in immature embryos. However,activity toward acyl CoA substrates or other synthetic substrates, forexample, is also contemplated herein.

[0055] Other plant kappa hydroxylases are obtainable from the specificexemplified sequences provided herein. Furthermore, it will be apparentthat one can obtain natural and synthetic plant kappa hydroxylasesincluding modified amino acid sequences and starting materials forsynthetic-protein modeling from the exemplified plant kappa hydroxylaseand from plant kappa hydroxylases which are obtained through the use ofsuch exemplified sequences. Modified amino acid sequences includesequences which have been mutated, truncated, increased and the like,whether such sequences were partially or wholly synthesized. Sequenceswhich are actually purified from plant preparations or are identical orencode identical proteins thereto, regardless of the method used toobtain the protein or sequence, are equally considered naturallyderived.

[0056] Thus, one skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared and used to screen and recover “homologous” or “related” kappahydroxylases from a variety of plant sources. Typically, nucleic acidprobes are labeled to allow detection, preferably with radioactivityalthough enzymes or other methods may also be used. For immunologicalscreening methods, antibody preparations either monoclonal or polyclonalare utilized. Polyclonal antibodies, although less specific, typicallyare more useful in gene isolation. For detection, the antibody islabeled using radioactivity or any one of a variety of secondantibody/enzyme conjugate systems that are commercially available.

[0057] Homologous sequences are found when there is an identity ofsequence and may be determined upon comparison of sequence information,nucleic acid or amino acid, or through hybridization reactions between aknown kappa hydroxylase and a candidate source. Conservative changes,such as Glu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also beconsidered in determining sequence homology. Typically, a lengthynucleic acid sequence may show as little as 50-60% sequence identity,and more preferably at least about 70% sequence identity, between thetarget sequence and the given plant kappa hydroxylase of interestexcluding any deletions which may be present, and still be consideredrelated. Amino acid sequences are considered homologous by as little as25% sequence identity between the two complete mature proteins. (Seegenerally, Doolittle, R. F., OF URFS and ORFS, University Science Books,CA, 1986.)

[0058] A genomic or other appropriate library prepared from thecandidate plant source of interest may be probed with conservedsequences from the plant kappa hydroxylase to identify homologouslyrelated sequences. Use of an entire cDNA or other sequence may beemployed if shorter probe sequences are not identified. Positive clonesare then analyzed by restriction enzyme digestion and/or sequencing.When a genomic library is used, one or more sequences may be identifiedproviding both the coding region, as well as the transcriptionalregulatory elements of the kappa hydroxylase gene from such plantsource. Probes can also be considerably shorter than the entiresequence. oligonucleotides may be used, for example, but should be atleast about 10, preferably at least about 15, more preferably at least20 nucleotides in length. When shorter length regions are used forcomparison, a higher degree of sequence identity is required than forlonger sequences. Shorter probes are often particularly useful forpolymerase chain reactions (PCR) , especially when highly conservedsequences can be identified (See Gould, et al., 1989 for examples of theuse of PCR to isolate homologous genes from taxonomically diversespecies).

[0059] When longer nucleic acid fragments are employed (>100 bp) asprobes, especially when using complete or large cDNA sequences, onewould screen with low stringencies (for example, 40-50° C. below themelting temperature of the probe) in order to obtain signal from thetarget sample with 20-50% deviation, i.e., homologous sequences. (Beltz,et al. 1983).

[0060] In a preferred embodiment, a plant kappa hydroxylase of thisinvention will have at least 60% overall amino acid sequence similaritywith the exemplified plant kappa hydroxylase. In particular, kappahydroxylases which are obtainable from an amino acid or nucleic acidsequence of a castor or lesquerella kappa hydroxylase are especiallypreferred. The plant kappa hydroxylases may have preferential activitytoward longer or shorter chain fatty acyl substrates. Plant fatty acylhydroxylases having oleate-12-hydroxylase activity andeicosenoate-14-hydroxylase activity are both considered homologouslyrelated proteins because of in vitro evidence (Howling et al., 1972),and evidence disclosed herein, that the castor kappa hydroxylase willact on both substrates. Hydroxylated fatty acids may be subject tofurther enzymatic modification by other enzymes which are normallypresent or are introduced by genetic engineering methods. For example,14-hydroxy-11,17-eicosadienoic acid, which is present in someLesquerella species (Smith 1985), is thought to be produced bydesaturation of 14-hydroxy-11-eicosenoic acid.

[0061] Again, not only can gene clones and materials derived thereof beused to identify homologous plant fatty acyl hydroxylases, but theresulting sequences obtained therefrom may also provide a further methodto obtain plant fatty acyl hydroxylases from other plant sources. Inparticular, PCR may be a useful technique to obtain related plant fattyacyl hydroxylases from sequence data provided herein. One skilled in theart will be able to design oligonucleotide probes based upon sequencecomparisons or regions of typically highly conserved sequence. Ofspecial interest are polymerase chain reaction primers based on theconserved regions of amino acid sequence between the castor kappahydroxylase and the L. fendleri hydroxylase (SEQ ID NO:4). Detailsrelating to the design and methods for a PCR reaction using these probesare described more fully in the examples.

[0062] It should also be noted that the fatty acyl hydroxylases of avariety of sources can be used to investigate fatty acid hydroxylationevents in a wide variety of plant and in vivo applications. Because allplants synthesize fatty acids via a common metabolic pathway, the studyand/or application of one plant fatty acid hydroxylase to a heterologousplant host may be readily achieved in a variety of species.

[0063] Once the nucleic acid sequence is obtained, the transcription, ortranscription and translation (expression), of the plant fatty acylhydroxylases in a host cell is desired to produce a ready source of theenzyme and/or modify the composition of fatty acids found therein in theform of free fatty acids, esters (particularly esterified toglyceroliplds or as components of wax esters), estolides, or ethers.Other useful applications may be found when the host cell is a planthost cell, in vitro and in vivo. For example, by increasing the amountof an kappa hydroxylase available to the plant, an increased percentageof ricinoleate or lesqueroleate (14-hydroxy-11-eicosenoic acid) may beprovided.

[0064] Kapoa Hydroxylase

[0065] By this invention, a mechanism for the biosynthesis of ricinoleicacid in plants is demonstrated. Namely, that a specific plant kappahydroxylase having preferential activity toward fatty acyl substrates isinvolved in the accumulation of hydroxylated fatty acids in at leastsome plant species. The use of the terms ricinoleate or ricinoleic acid(or lesqueroleate or lesquerolic acid, densipoleate etc.) is intended toinclude the free acids, the ACP and CoA esters, the salts of theseacids, the glycerolipid esters (particularly the triacylglycerolesters), the wax esters, the estolides and the ether derivatives ofthese acids.

[0066] The determination that plant fatty acyl hydroxylases are activein the in vivo production of hydroxylated fatty acids suggests severalpossibilities for plant enzyme sources. In fact, hydroxylated fattyacids are found in some natural plant species in abundance. For example,three hydroxy fatty acids related to ricinoleate occur in major amountsin seed oils from various Lesquerella species. Of particular interest,lesquerolic acid is a 20 carbon homolog of ricinoleate with twoadditional carbons at the carboxyl end of the chain (Smith 1985). Othernatural plant sources of hydroxylated fatty acids include but are notlimited to seeds of the Linum genus, seeds of Wrightia species,Lycopodium species, Strophanthus species, Convolvulaces species,Calendula species and many others (van de Loo et al., 1993).

[0067] Plants having significant presence of ricinoleate orlesqueroleate or desaturated other or modified derivatives of thesefatty acids are preferred candidates to obtain naturally-derived kappahydroxylases. For example, Lesquerella densipila contains adiunsaturated 18 carbon fatty acid with a hydroxyl group (van de Loo etal., 1993) that is thought to be produced by an enzyme that is closelyrelated to the castor kappa hydroxylase, according to the theory onwhich this invention is based. In addition, a comparison between kappahydroxylases and between plant fatty acyl hydroxylases which introducehydroxyl groups at positions other than the 12-carbon of oleate or the14-carbon of lesqueroleate or on substrates other than oleic acid andicosenoic acid may yield insights for protein modeling or othermodifications to create synthetic hydroxylases as discussed above. Forexample, on the basis of information gained from structural comparisonsof the Δ12 desaturases and the kappa hydroxylase, we envision makinggenetic modifications in the structural genes for Δ12 desaturases thatconvert these desaturases to kappa-hydroxylases. We also envision makingchanges in Δ15 hydroxylases that convert these to hydroxylases withcomparable substrate specificity to the desaturases (e.g., conversion of18:2^(Δ9,12) to 15OH-18:2^(Δ9,12). Since the difference between ahydroxylase and a desaturases concerns the disposition of one proton, weenvision that by systematically changing the charged groups in theregion of the enzyme near the active site, we can effect this change.

[0068] Especially of interest are fatty acyl hydroxylases whichdemonstrate activity toward fatty acyl substrates other than oleate, orwhich introduce the hydroxyl group at a location other than the C12carbon. As described above, other plant sources may also provide sourcesfor these enzymes through the use of protein purification, nucleic acidprobes, antibody preparations, protein modeling, or sequencecomparisons, for example, and of special interest are the respectiveamino acid and nucleic acid sequences corresponding to such plant fattyacyl hydroxylases. Also as previously described, once a nucleic acidsequence is obtained for the given plant hydroxylase, further plantsequences may be compared and/or probed to obtain homologously relatedDNA sequences thereto and so on.

[0069] Genetic Engineering Applications

[0070] As is well known in the art, once a cDNA clone encoding a plantkappa hydroxylase is obtained, it may be used to obtain itscorresponding genomic nucleic acid sequences thereto.

[0071] The nucleic acid sequences which encode plant kappa hydroxylasesmay be used in various constructs, for example, as probes to obtainfurther sequences from the same or other species. Alternatively, thesesequences may be used in conjunction with appropriate regulatorysequences to increase levels of the respective hydroxylase of interestin a host cell for the production of hydroxylated fatty acids or studyof the enzyme in vitro or in vivo or to decrease or increase levels ofthe respective hydroxylase of interest for some applications when thehost cell is a plant entity, including plant cells, plant parts(including but not limited to seeds, cuttings or tissues) and plants.

[0072] A nucleic acid sequence encoding a plant kappa hydroxylase ofthis invention may include genomic, cDNA or mRNA sequence. By “encoding”is meant that the sequence corresponds to a particular amino acidsequence either in a sense or anti-sense orientation. By “recombinant”is meant that the sequence contains a genetically engineeredmodification through manipulation via mutagenesis, restriction enzymes,and the like. A cDNA sequence may or may not encode pre-processingsequences, such as transit or signal peptide sequences. Transit orsignal peptide sequences facilitate the delivery of the protein to agiven organelle and are frequently cleaved from the polypeptide uponentry into the organelle, releasing the “mature” sequence. The use ofthe precursor DNA sequence is preferred in plant cell expressioncassettes.

[0073] Furthermore, as discussed above the complete genomic sequence ofthe plant kappa hydroxylase may be obtained by the screening of agenomic library with a probe, such as a cDNA probe, and isolating thosesequences which regulate expression in seed tissue. In this manner, thetranscription and translation initiation regions, introns, and/ortranscript termination regions of the plant kappa hydroxylase may beobtained for use in a variety of DNA constructs, with or without thekappa hydroxylase structural gene. Thus, nucleic acid sequencescorresponding to the plant kappa hydroxylase of this invention may alsoprovide signal sequences useful to direct transport into an organelle 5′upstream non-coding regulatory regions (promoters) having useful tissueand timing profiles, 3′ downstream non-coding regulatory region usefulas transcriptional and translational regulatory regions and may lendinsight into other features of the gene.

[0074] Once the desired plant kappa hydroxylase nucleic acid sequence isobtained, it may be manipulated in a variety of ways. Where the sequenceinvolves non-coding flanking regions, the flanking regions may besubjected to resection, mutagenesis, etc. Thus, transitions,transversions, deletions, and insertions may be performed on thenaturally occurring sequence. In addition, all or part of the sequencemay be synthesized. In the structural gene, one or more codons may bemodified to provide for a modified amino acid sequence, or one or morecodon mutations may be introduced to provide for a convenientrestriction site or other purpose involved with construction orexpression. The structural gene may be further modified by employingsynthetic adapters, linkers to introduce one or more convenientrestriction sites, or the like.

[0075] The nucleic acid or amino acid sequences encoding a plant kappahydroxylase of this invention may be combined with other non-native, or“heterologous”, sequences in a variety of ways. By “heterologous”sequences is meant any sequence which is not naturally found joined tothe plant kappa hydroxylase, including, for example, combination ofnucleic acid sequences from the same plant which are not naturally foundjoined together.

[0076] The DNA sequence encoding a plant kappa hydroxylase of thisinvention may be employed in conjunction with all or part of the genesequences normally associated with the kappa hydroxylase. In itscomponent parts, a DNA sequence encoding kappa hydroxylase is combinedin a DNA construct having, in the 5′ to 3′ direction of transcription, atranscription initiation control region capable of promotingtranscription and translation in a host cell, the DNA sequence encodingplant kappa hydroxylase and a transcription and translation terminationregion.

[0077] Potential host cells include both prokaryotic and eukaryoticcells. A host cell may be unicellular or found in a multicellulardifferentiated or undifferentiated organism depending upon the intendeduse. Cells of this invention may be distinguished by having a plantkappa hydroxylase foreign to the wild-type cell present therein, forexample, by having a recombinant nucleic acid construct encoding a plantkappa hydroxylase therein.

[0078] Depending upon the host, the regulatory regions will vary,including regions from viral, plasmid or chromosomal genes, or the like.For expression in prokaryotic or eukaryotic microorganisms, particularlyunicellular hosts, a wide variety of constitutive or regulatablepromoters may be employed. Expression in a microorganism can provide aready source of the plant enzyme. Among transcriptional initiationregions which have been described are regions from bacterial and yeasthosts, such as E. coli, B. subtilis, Saccharomyces cerevisiae, includinggenes such as beta-galactosidase, T7 polymerase, tryptophan E and thelike.

[0079] For the most part, the constructs will involve regulatory regionsfunctional in plants which provide for modified production of plantkappa hydroxylase with resulting modification of the fatty acidcomposition. The open reading frame, coding for the plant kappahydroxylase or functional fragment thereof will be joined at its 5′ endto a transcription initiation regulatory region such as the wild-typesequence naturally found 5′ upstream to the kappa hydroxylase structuralgene. Numerous other transcription initiation regions are availablewhich provide for a wide variety of constitutive or regulatable, e.g.,inducible, transcription of the structural gene functions.

[0080] Among transcriptional initiation regions used for plants are suchregions associated with the structural genes such as for nopaline andmannopine synthases, or with napin, soybean β-conglycinin, oleosin, 12Sstorage protein, the cauliflower mosaic virus 35S promoters and thelike. The transcription/translation initiation regions corresponding tosuch structural genes are found immediately 5′ upstream to therespective start codons.

[0081] In embodiments wherein the expression of the kappa hydroxylaseprotein is desired in a plant host, the use of all or part of thecomplete plant kappa hydroxylase gene is desired; namely all or part ofthe 5′ upstream non-coding regions (promoter) together with thestructural gene sequence and 3′ downstream non-coding regions may beemployed. If a different promoter is desired, such as a promoter nativeto the plant host of interest or a modified promoter, i.e., havingtranscription initiation regions derived from one gene source andtranslation initiation regions derived from a different gene source,including the sequence encoding the plant kappa hydroxylase of interest,or enhanced promoters, such as double 35S CaMV promoters, the sequencesmay be joined together using standard techniques.

[0082] For such applications when 5′ upstream non-coding regions areobtained from other genes regulated during seed maturation, thosepreferentially expressed in plant embryo tissue, such as transcriptioninitiation control regions from the D. napus napin gene, or theArabidopsis 12S storage protein, or soybean β-conglycinin (Bray et al.,1987), or the L. fendleri kappa hydroxylase promoter described hereinare desired. Transcription initiation regions which are preferentiallyexpressed in seed tissue, i.e., which are undetectable in other plantparts, are considered desirable for fatty acid modifications in order tominimize any disruptive or adverse effects of the gene product.

[0083] Regulatory transcript termination regions may be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding the plant kappa hydroxylase ora convenient transcription termination region derived from a differentgene source, for example, the transcript termination region which isnaturally associated with the transcript initiation region. Where thetranscript termination region is from a different gene source, it willcontain at least about 0.5 kb, preferably about 1-3 kb of sequence 3′ tothe structural gene from which the termination region is derived.

[0084] Plant expression or transcription constructs having a plant kappahydroxylase as the DNA sequence of interest for increased or decreasedexpression thereof may be employed with a wide variety of plant life,particularly, plant life involved in the production of vegetable oilsfor edible and industrial uses. Most especially preferred are temperateoilseed crops. Plants of interest include, but are not limited torapeseed (Canola and high erucic acid varieties), Crambe, Brassicajuncea, Brassica nigra, meadowfoam, flax, sunflower, safflower, cotton,Cuphea, soybean, peanut, coconut and oil palms and corn. An importantcriterion in the selection of suitable plants for the introduction onthe kappa hydroxylase is the presence in the host plant of a suitablesubstrate for the hydroxylase. Thus, for example, production ofricinoleic acid will be best accomplished in plants that normally havehigh levels of oleic acid in seed lipids. Similarly, production oflesquerolic acid will best be accomplished in plants that have highlevels of icosenoic acid in seed lipids.

[0085] Depending on the method for introducing the recombinantconstructs into the host cell, other DNA sequences may be required.Importantly, this invention is applicable to dicotyledons andmonocotyledons species alike and will be readily applicable to newand/or improved transformation and regulation techniques. The method oftransformation is not critical to the current invention; various methodsof plant transformation are currently available. As newer methods areavailable to transform crops, they may be directly applied hereunder.For example, many plant species naturally susceptible to Agrobacteriuminfection may be successfully transformed via tripartite or binaryvector methods of Agrobacterium mediated transformation. In addition,techniques of microinjection, DNA particle bombardment, electroporationhave been developed which allow for the transformation of variousmonocot and dicot plant species.

[0086] In developing the DNA construct, the various components of theconstruct or fragments thereof will normally be inserted into aconvenient cloning vector which is capable of replication in a bacterialhost, e.g., E. coli. Numerous vectors exist that have been described inthe literature. After each cloning, the plasmid may be isolated andsubjected to further manipulation, such as restriction, insertion of newfragments, ligation, deletion, insertion, resection, etc., so as totailor the components of the desired sequence. Once the construct hasbeen completed, it may then be transferred to an appropriate vector forfurther manipulation in accordance with the manner of transformation ofthe host cell.

[0087] Normally, included with the DNA construct will be a structuralgene having the necessary regulatory regions for expression in a hostand providing for selection of transformant cells. The gene may providefor resistance to a cytotoxic agent, e.g., antibiotic, heavy metal,toxin, etc., complementation providing prototropy to an auxotrophichost, viral immunity or the like. Depending upon the number of differenthost species the expression construct or components thereof areintroduced, one or more markers may be employed, where differentconditions for selection are used for the different hosts.

[0088] It is noted that the degeneracy of the DNA code provides thatsome codon substitutions are permissible of DNA sequences without anycorresponding modification of the amino acid sequence.

[0089] As mentioned above, the manner in which the DNA construct isintroduced into the plant host is not critical to this invention. Anymethod which provides for efficient transformation may be employed.Various methods for plant cell transformation include the use of Ti- orRi-plasmids, microinjection, electroporation, infiltration, imbibition,DNA particle bombardment, liposome fusion, DNA bombardment or the like.In many instances, it will be desirable to have the construct borderedon one or both sides of the T-DNA, particularly having the left andright borders, more particularly the right border. This is particularlyuseful when the construct uses A. tumefaciens or A. rhizogenes as a modefor transformation, although the T-DNA borders may find use with othermodes of transformation.

[0090] Where Agrobacterium is used for plant cell transformation, avector may be used which may be introduced into the Agrobacterium hostfor homologous recombination with T-DNA or the Ti- or Ri-plasmid presentin the Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNAfor recombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall), the latter being permissible, solong as the vir genes are present in the transformed Agrobacterium host.The armed plasmid can give a mixture of normal plant cells and gall.

[0091] In some instances where Agrobacterium is used as the vehicle fortransforming plant cells, the expression construct bordered by the T-DNAborder(s) will be inserted into a broad host spectrum vector, therebeing broad host spectrum vectors described in the literature. Commonlyused is pRK2 or derivatives thereof. See, for example, Ditta et al.,(1980), which is incorporated herein by reference. Included with theexpression construct and the T-DNA will be one or more markers, whichallow for selection of transformed Agrobacterium and transformed plantcells. A number of markers have been developed for use with plant cells,such as resistance to kanamycin, the aminoglycoside G418, hygromycin, orthe like. The particular marker employed is not essential to thisinvention, one or another marker being preferred depending on theparticular host and the manner of construction.

[0092] For transformation of plant cells using Agrobacterium, explantsmay be combined and incubated with the transformed Agrobacterium forsufficient time for transformation, the bacteria killed, and the plantcells cultured in an appropriate selective medium. Once callus forms,shoot formation can be encouraged by employing the appropriate planthormones in accordance with known methods and the shoots transferred torooting medium for regeneration of plants. The plants may then be grownto seed and the seed used to establish repetitive generations and forisolation of vegetable oils.

[0093] Using Hydroxylase Genes to Alter the Activity of Fatty AcidDesaturases

[0094] A widely acknowledged goal of current efforts to improve thenutritional quality of edible plant oils, or to facilitate industrialapplications of plant oils, is to alter the level of desaturation ofplant storage lipids (Topfer et al., 1995). In particular, in many cropspecies it is considered desirable to reduce the level ofpolyunsaturation of storage lipids and to increase the level of oleicacid. The precise amount of the various fatty acids in a particularplant oil varies with the intended application. Thus, it is desirable tohave a robust method that will permit genetic manipulation of the levelof unsaturation to any desired level.

[0095] Substantial progress has recently been made in the isolation ofgenes encoding plant fatty acid desaturases (reviewed in Topfer et al.,1995). These genes have been introduced into various plant species andused to alter the level of fatty acid unsaturation in one of three ways.First, the genes can be placed under transcriptional control of a strongpromoter so that the amount of the corresponding enzyme is increased. Insome cases this leads to an increase in the amount of the fatty acidthat is the product of the reaction catalyzed by the enzyme. Forexample, Arondel et al. (1992) increased the amount of linolenic acid(18:3) in tissues of transgenic Arabidopsis plants by placing theendoplasmic reticulum-localized fad3 gene under transcriptional controlof the strong constitutive cauliflower mosaic virus 35S promoter.

[0096] A second method of using cloned genes to alter the level of fattyacid unsaturation is to cause transcription of all or part of a gene intransgenic tissues so that the transcripts have an antisense orientationrelative to the normal mode of transcription. This has been used by anumber of laboratories to reduce the level of expression of one or moredesaturase genes that have significant nucleotide sequence homology tothe gene used in the construction of the antisense gene (reviewed inTopfer et al.). For instance, antisense repression of the oleateΔ12-desaturase in transgenic rapeseed resulted in a strong increase inoleic acid content (cf., Topfer et al., 1995).

[0097] A third method for using cloned genes to alter fatty aciddesaturation is to exploit the phenomenon of cosuppression or“gene-silencing” (Matzke et al., 1995). Although the mechanismsresponsible for gene silencing are not known in any detail, it hasfrequently been observed that in transgenic plants, expression of anintroduced gene leads to inactivation of homologous endogenous genes.

[0098] For example, high-level sense expression of the Arabidopsis fad8gene, which encodes a chloroplast-localized Δ15-desaturase, intransgenic Arabidopsis plants caused suppression of the endogenous copyof the fad8 gene and the homologous fad7 gene (which encodes an isozymeof the fad8 gene) (Gibson et al., 1994). The fad7 and fad8 genes areonly 76% identical at the nucleotide level. At the time of publication,this example represented the most divergent pair of plant genes forwhich cosuppression had been observed.

[0099] In view of previous evidence concerning the relatively high levelof nucleotide sequence homology required to obtain cosuppression, it isnot obvious to one skilled in the art that sense expression intransgenic plants of the castor fatty acyl hydroxylase of this inventionwould significantly alter the amount of unsaturation of storage lipids.

[0100] However, we have established that fatty acyl hydroxylase genescan be used for this purpose as taught in Example 4 of this invention.Of particular importance, this invention teaches the use of fatty acylhydroxylase genes to increase the proportion of oleic acid in transgenicplant tissues. The mechanism by which expression of the gene exerts thiseffect is not known but may be due to one of several possibilities whichare elaborated upon in Example 4.

[0101] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded for purposes of illustration only and are not intended to limitthe present invention.

EXAMPLES

[0102] In the experimental disclosure which follows, all temperaturesare given in degrees centigrade (°), weights are given in grams (g),milligram (mg) or micrograms (μg), concentrations are given as molar(M), millimolar (mM) or micromolar (μM) and all volumes are given inliters (l), microliters (μl) or milliliters (ml), unless otherwiseindicated.

Example 1 Production of Novel Hydroxylated Fatty Acids in Arabidopsisthaliana

[0103] Overview

[0104] The kappa hydroxylase encoded by the previously described fah12gene from Castor (U.S. patent application Ser. No. 08/320,982) was usedto produce ricinoleic acid, lesquerolic acid, densipolic acid andauricolic acid in transgenic Arabidopsis plants. This examplespecifically discloses the method taught in Example 2 of U.S. patentapplication Ser. No. 08/320,982.

[0105] Production of Transgenic Plants

[0106] A variety of methods have been developed to insert a DNA sequenceof interest into the genome of a plant host to obtain the transcriptionand translation of the sequence to effect phenotypic changes. Thefollowing methods represent only one of many equivalent means ofproducing transgenic plants and causing expression of the hydroxylasegene.

[0107] Arabidopsis plants were transformed, by Agrobacterium-mediatedtransformation, with the kappa hydroxylase encoded by the Castor fah12gene on binary Ti plasmid pB6. This plasmid was previously used totransform Nicotiana tabacum for the production of ricinoleic acid (U.S.patent application Ser. No. 08/320,982).

[0108] Inoculums of Agrobacterium tumefaciens strain GV3101 containingbinary Ti plasmid pB6 were plated on L-broth plates containing 50 μg/mlkanamycin and incubated for 2 days at 30° C. Single colonies were usedto inoculate large liquid cultures (L-broth medium with 50 mg/lrifampicin, 110 mg/l gentamycin and 200 mg/l kanamycin) to be used forthe transformation of Arabidopsis plants.

[0109] Arabidopsis plants were transformed by the in plantatransformation procedure essentially as described by Bechtold et al.,(1993). Cells of A. tumefaciens GV3101(pB6) were harvested from liquidcultures by centrifugation, then resuspended in infiltration medium atOD₆₀₀=0.8 (Infiltration medium was Murashige and Skoog macro andmicronutrient medium (Sigma Chemical Co., St. Louis, Mo.) containing 10mg/l 6-benzylaminopurine and 5% glucose). Batches of 12-15 plants weregrown for 3 to 4 weeks in natural light at a mean daily temperature ofapproximately 25° C. in 3.5 inch pots containing soil. The intact plantswere immersed in the bacterial suspension then transferred to a vacuumchamber and placed under 600 mm of vacuum produced by a laboratoryvacuum pump until tissues appeared uniformly water-soaked (approximately10 min). The plants were grown at 25° C. under continuous light (100μmol m⁻² s⁻¹ irradiation in the 400 to 700 nm range) for four weeks. Theseeds obtained from all the plants in a pot were harvested as one batch.The seeds were sterilized by sequential treatment for 2 min with ethanolfollowed by 10 min in a mixture of household bleach (Chlorox), water andTween-80 (50%, 50%, 0.05%) then rinsed thoroughly with sterile water.The seeds were plated at high density (2000 to 4000 per plate) ontoagar-solidified medium in 100 mm petri plates containing 1/2×Murashigeand Skoog salts medium enriched with B5 vitamins (Sigma Chemical Co.,St. Louis, Mo.) and containing kanamycin at 50 mg/l. After incubationfor 48 h at 4° C. to stimulate germination, seedlings were grown for aperiod of seven days until transformants were clearly identifiable ashealthy green seedlings against a background of chlorotickanamycin-sensitive seedlings. The transformants were transferred tosoil for two weeks before leaf tissue could be used for DNA and lipidanalysis. More than 20 transformants were obtained.

[0110] DNA was extracted from young leaves from transformants to verifythe presence of an intact fah12 gene. The presence of the transgene in anumber of the putative transgenic lines was verified by using thepolymerase chain reaction to amplify the insert from pB6. The primersused were HF2=GCTCTTTTGTGCGCTCATTC (SEQ ID NO:12) andHR1=CGGTACCAGAAAACGCCTTG (SEQ ID NO:13), which were designed to allowthe amplification of a 700 bp fragment. Approximately 100 ng of genomicDNA was added to a solution containing 25 pmol of each primer, 1.5 U Taqpolymerase (Boehringer Manheim), 200 uM of dNTPs, 50 mM KCl, 10 mMTris.Cl (pH 9), 0.1% (v/v) Triton X-100, 1.5 mM MgCl₂, 3% (v/v)formamide, to a final volume of 50 μl. Amplifications conditions were: 4min denaturation step at 94° C., followed by 30 cycles of 92° C. for 1min, 55° C. for 1 min, 72° C. for 2 min. A final extension step closedthe program at 72° C. for 5 min. Transformants could be positivelyidentified after visualization of a characteristic 1 kb amplifiedfragment on an ethidium bromide stained agarose gel. All transgeniclines tested gave a PCR product of a size consistent with the expectedgenotype, confirming that the lines were, indeed, transgenic. Allfurther experiments were done with three representative transgenic linesof the wild type designated as 1-3, 4D, 7-4 and one transgenic line ofthe fad2 mutant line JB12. The transgenic JB12 line was included inorder to test whether the increased accumulation of oleic acid in thismutant would have an effect on the amount of ricinoleic acid thataccumulated in the transgenic plants.

[0111] Analysis of Transgenic Plants

[0112] Leaves and seeds from fah12 transgenic Arabidopsis plants wereanalyzed for the presence of hydroxylated fatty acids using gaschromatography. Lipids were extracted from 100-200 mg leaf tissue or 50seeds. Fatty acid methyl esters (FAMES) were prepared by placing tissuein 1.5 ml of 1.0 M methanolic HCl (Supelco Co.) in a 13×100 mm glassscrew-cap tube capped with a teflon-lined cap and heated to 80° C. for 2hours. Upon cooling, 1 ml petroleum ether was added and the FAMESremoved by aspirating off the ether phase which was then dried under anitrogen stream in a glass tube. One hundred μl ofN,O-bis(Trimethylsilyl)trifluoroacetamide (BSTFA; Pierce Chemical Co)and 200 μl acetonitrile was added to derivatize the hydroxyl groups. Thereaction was carried out at 70° C. for 15 min. The products were driedunder nitrogen, redissolved in 100 μl chloroform and transferred to agas chromatograph vial. Two μl of each sample were analyzed on a SP2340fused silica capillary column (30 m, 0.75 mm ID, 0.20 mm film, Supelco),using a Hewlett-Packard 5890 II series Gas Chromatograph. The sampleswere not split, the temperature program was 195° C. for 18 min,increased to 230° C. at 25° C./min, held at 230° C. for 5 min then downto 195° C. at 25° C./min., and flame ionization detectors were used.

[0113] The chromatographic elution time of methyl esters and O-TMSderivatives of ricinoleic acid, lesquerolic acid and auricolic acid wasestablished by GC-MS of lipid samples from seeds of L. fendleri andcomparison to published chromatograms of fatty acids from this species(Carlson et al., 1990). A O-TMS-methyl-ricinoleate standard was preparedfrom ricinoleic acid obtained from Sigma Chemical Co (St, Louis, Mo.).O-TMS-methyl-lesqueroleate and O-TMS-methyl-auricoleate standards wereprepared from triacylglycerols purified from seeds of L. fendleri. Themass spectrum of O-TMS-methyl-ricinoleate, O-TMS-methyl-densipoleate,O-TMS-methyl-lesqueroleate, and O-TMS-methyl-auricoleate are shown inFIGS. 1A-D, respectively. The structures of the characteristic ionsproduced during mass spectrometry of these derivatives are shown in FIG.2.

[0114] Lipid extracted from transgenic tissues were analyzed by gaschromatography and mass spectrometry for the presence of hydroxylatedfatty acids. As a matter of reference, the average fatty acidcomposition of leaves in Arabidopsis wild type and fad2 mutant lines wasreported by Miquel and Browse (1992). Gas chromatograms of methylatedand silylated fatty acids from seeds of wild type and a fah12 transgenicwild type plant are shown in FIGS. 3A and 3B, respectively. The profilesare very similar except for the presence of three small but distinctpeaks at 14.3, 15.9 and 18.9 minutes. A very small peak at 20.15 min wasalso evident. The elution time of the peaks at 14.3 and 18.9 mincorresponded precisely to that of comparably prepared ricinoleic andlesquerolic standards, respectively. No significant differences wereobserved in lipid extracts from leaves or roots of the wild type and thefah12 transgenic wild type lines (Table 1). Thus, in spite of the factthat the fah12 gene is expressed throughout the plant, we observedeffects on fatty acid composition only in seed tissue. A similarobservation was described previously for transgenic fah12 tobacco inpatent application Ser. No. 08/320,982. TABLE 1 Fatty acid compositionof lipids from transgenic and wild type Arabidopsis. The values are themeans obtained from analysis of samples from three independenttransgenic lines, or three independent samples of wild type and fad2lines. Seed Leaf Root Fatty FAH12/ FAH12/ FAH12/ FAH12/ acid WT WT fad2JB12 WT WT WT WT 16:0 8.5 8.2 6.4 6.1 16.5 17.5 23.9 24.9 16:3 0 0 0 010.1 9.8 0 0 18:0 3.2 3.5 2.9 3.5 1.3 1.2 2.0 1.9 18:1 15.4 26.3 43.447.8 2.4 3.4 5.4 3.2 18:2 27.0 21.4 10.2 7.2 15.1 14.0 32.2 29.4 18:322.0 16.6 — 9.7 36.7 36.0 26.7 30.6 20:1 14.0 14.3 — 13.1 0 0 0 0 22:12.0 1.0 0.5 0.5 0 0 0 0 24:1 2.5 1.7 2.0 1.6 0 0 0 0 18:1- 0 0.4 0.3 0 00 0 0 OH 18:2- 0 0.4 0.3 0 0 0 0 0 OH 20:1- 0 0.2 0.1 0 0 0 0 0 OH 20:2-0 0.1 0.1 0 0 0 0 0 OH

[0115] In order to confirm that the observed new peaks in the transgeniclines corresponded to derivatives of ricinoleic, lesquerolic, densipolicand auricolic acids, mass spectrometry was used. The fatty acidderivatives were resolved by gas chromatography as described aboveexcept that a Hewlett-Packard 5971 series mass selective detector wasused in place of the flame ionization detector used in the previousexperiment. The spectra of the four new peaks in FIG. 3B (peak numbers10, 11, 12 and 13) are shown in FIGS. 4A-D, respectively. Comparison ofthe spectrum obtained for the standards with that obtained for the fourpeaks from the transgenic lines confirms the identity of the four newpeaks. On the basis of the three characteristic peaks at M/Z 187, 270and 299, peak 10 is unambiguously identified as O-TMS-methylricinoleate.On the basis of the three characteristic peaks at M/Z 185, 270 and 299,peak 11 is unambiguously identified as O-TMS-methyldensipoleate. On thebasis of the three characteristic peaks at M/Z 187, 298 and 327, peak 12is unambiguously identified as O-TMS-methyllesqueroleate. On the basisof the three characteristic peaks at M/Z 185, 298 and 327, peak 13 isunambiguously identified as O-TMS-methylauricoleate.

[0116] These results unequivocally demonstrate the identity of the fah12cDNA as encoding a hydroxylase that hydroxylates both oleic acid toproduce ricinoleic acid and also hydroxylates icosenoic acid to producelesquerolic acid. These results also provide additional evidence thatthe hydroxylase can be functionally expressed in a heterologous plantspecies in such a way that the enzyme is catalytically functional. Theseresults also demonstrate that expression of this hydroxylase gene leadsto accumulation of ricinoleic, lesquerolic, densipolic and auricolicacids in a plant species that does not normally accumulate hydroxylatedfatty acids in extractable lipids.

[0117] The presence of lesquerolic acid in the transgenic plants wasanticipated in the previous patent application (Ser. No. 08/320,982)based on the biochemical evidence suggesting broad substrate specificityof the kappa hydroxylase. By contrast, the accumulation of densipolicand auricolic acids was less predictable. Since Arabidopsis does notnormally contain significant quantities of the non-hydroxylatedprecursors of these fatty acids which could serve as substrates for thehydroxylase, it appears that one or more of the three n-3 fatty aciddesaturases known in Arabidopsis (eg., fad3, fad7, fad8; reviewed inGibson et al., 1995) are capable of desaturating the hydroxylatedcompounds at the n-3 position. That is, densipolic acid is produced bythe action of an n-3 desaturase on ricinoleic acid. Auricolic acid isproduced by the action of an n-3 desaturase on lesquerolic acid. Becauseit is located in the endoplasmic reticulum, the fad3 desaturase isalmost certainly responsible. This can be tested in the future byproducing fah12-containing transgenic plants of the fad3-deficientmutant of Arabidopsis (similar experiments can be done with fad7 andfad8). It is also formally possible that the enzymes that normallyelongate 18:1^(cisΔ9) to 20:1^(cisΔ11) may elongate 12OH-18:1^(cisΔ9) to14OH-20:1^(cisΔ11), and 12OH-18:2^(cisΔ9, 15) to 14OH-20:2^(cisΔ11, 17).

[0118] The amount of the various fatty acids in seed, leaf and rootlipids of the control and transgenic plants is also presented inTable 1. Although the amount of hydroxylated fatty acids produced inthis example is less than desired for commercial production ofricinoleate and other hydroxylated fatty acids from plants, we envisionnumerous improvements of this invention that will increase the level ofaccumulation of hydroxylated fatty acids in plants that express thefah12 or related hydroxylase genes. Improvements in the level and tissuespecificity of expression of the hydroxylase gene is envisioned. Methodsto accomplish this by the use of strong, seed-specific promoters such asthe B. napus napin promoter or the native promoters of the castor fah12gene or the corresponding hydroxylase gene from L. fendleri will beobvious to one skilled in the art. Additional improvements areenvisioned to involve modification of the enzymes which cleavehydroxylated fatty acids from phosphatidylcholine, reduction in theactivities of enzymes which degrade hydroxylated fatty acids andreplacement of acyltransferases which transfer hydroxylated fatty acidsto the sn-1, sn-2 and sn-3 positions of glycerolipids. Although genesfor these enzymes have not been described in the scientific literature,their utility in improving the level of production of hydroxylated fattyacids can be readily envisioned based on the results of biochemicalinvestigations of ricinoleate synthesis.

[0119] Although Arabidopsis is not an economically important plantspecies, it is widely accepted by plant biologists as a model for higherplants. Therefore, the inclusion of this example is intended todemonstrate the general utility of the invention described here and inthe previous application (Ser. No. 08/320,982) to the modification ofoil composition in higher plants. One advantage of studying theexpression of this novel gene in Arabidopsis is the existence in thissystem of a large body of knowledge on lipid metabolism, as well as theavailability of a collection of mutants which can be used to provideuseful information on the biochemistry of fatty acid hydroxylation inplant species. Another advantage is the ease of transposing any of theinformation obtained on metabolism of ricinoleate in Arabidopsis toclosely related species such as the crop plants Brassica napus, Brassicajuncea or Crambe abyssinica in order to mass produce ricinoleate,lesqueroleate or other hydroxylated fatty acids for industrial use. Thekappa hydroxylase is useful for the production of ricinoleate orlesqueroleate in any plant species that accumulates significant levelsof the precursors, oleic acid and icosenoic acid. Of particular interestare genetically modified varieties that accumulate high levels of oleicacid. Such varieties are currently available for sunflower and Canola.Production of lesquerolic acid and related hydroxy fatty acids can beachieved in species that accumulate high levels of icosenoic acid orother long chain monoenoic acids. Such plants may in the future beproduced by genetic engineering of plants that do not normally make suchprecursors. Thus, we envision that the use of the kappa hydroxylase isof general utility.

Example 2 Isolation of Lesquerella Kappa Hydroxylase Genomic Clone

[0120] Overview

[0121] Regions of nucleotide sequence that were conserved in both theCastor kappa hydroxylase and the Arabidopsis fad2 Δ12 fatty aciddesaturase were used to design oligonucleotide primers. These were usedwith genomic DNA from Lesquerella fendleri to amplify fragments ofseveral homologous genes. These amplified fragments were then used ashybridization probes to identify full length genomic clones from agenomic library of L. fendleri.

[0122] Hydroxylated fatty acids are specific to the seed tissue ofLesquerella sp., and are not found to any appreciable extent invegetative tissues. One of the two genes identified by this method wasexpressed in both leaves and developing seeds and is therefore thoughtto correspond to the Δ12 fatty acid desaturase. The other gene wasexpressed at high levels in developing seeds but was not expressed orwas expressed at very low levels in leaves and is the kappa hydroxylasefrom this species. The identity of the gene as a fatty acyl hydroxylasewas established by functional expression of the gene in yeast.

[0123] The identity of this gene will also be established by introducingthe gene into transgenic Arabidopsis plants and showing that it causesthe accumulation of ricinoleic acid, lesquerolic acid, densipolic acidand auricolic acid in seed lipids. The promoter of this gene is also ofutility because it is able to direct expression of a gene specificallyin developing seeds at a time when storage lipids are accumulating. Thispromoter is, therefore, of great utility for many applications in thegenetic engineering of seeds, particularly in members of theBrassicacea.

[0124] The various steps involved in this process are described indetail below. Unless otherwise indicated, routine methods formanipulating nucleic acids, bacteria and phage were as described bySambrook et al. (1989).

[0125] Isolation of a Fragment of the Lesguerella Kappa Hydroxylase Gene

[0126] oligonucleotide primers for the amplification of the L. fendlerikappa hydroxylase were designed by choosing regions of high deducedamino acid sequence homology between the Castor kappa hydroxylase andthe Arabidopsis Δ12 desaturase (fad2). Because most amino acids areencoded by several different codons, these oligonucleotides weredesigned to encode all possible codons that could encode thecorresponding amino acids.

[0127] The sequence of these mixed oligonucleotides was:

[0128] Oligo 1: TAYWSNCAYMGNMGNCAYCA (SEQ ID NO:14)

[0129] Oligo 2: RTGRTGNGCNACRTGNGTRTC (SEQ ID NO:15)

[0130] (Where: Y=C+T; W=A+T; S=G+C; N=A+G+C+T; M=A+C; R=A+G)

[0131] These oligonucleotides were used to amplify a fragment of DNAfrom L. fendleri genomic DNA by the polymerase chain reaction (PCR)using the following conditions: Approximately 100 ng of genomic DNA wasadded to a solution containing 25 pmol of each primer, 1.5 U Taqpolymerase (Boehringer Manheim), 200 uM of dNTPs, 50 mM KCl, 10 mMTris.Cl (pH 9), 0.1% (v/v) Triton X-100, 1.5 mM MgCl₂, 3% (v/v)formamide, to a final volume of 50 μl. Amplifications conditions were: 4min denaturation step at 94° C., followed by 30 cycles of 92° C. for 1min, 55° C. for 1 min, 72° C. for 2 min. A final extension step closedthe program at 72° C. for 5 min.

[0132] PCR products of approximately 540 bp were observed followingelectrophoretic separation of the products of the PCR reaction inagarose gels. Two of these fragments were cloned into pBluescript(Stratagene) to give rise to plasmids pLesq2 and pLesq3. The sequence ofthe inserts in these two plasmids was determined by the chaintermination method. The sequence of the insert in pLesq2 is presented asFIG. 5 (SEQ ID NO:1) and the sequence of the insert in pLesq3 ispresented as FIG. 6 (SEQ ID NO:2). The high degree of sequence identitybetween the two clones indicated that they were both potentialcandidates to be either a Δ12 desaturase or a gamma hydroxylase.

[0133] Northern Analysis

[0134] In L. fendleri, hydroxylated fatty acids are found in largeamounts in seed oils but are not found in appreciable amounts in leaves.An important criterion in discriminating between a fatty acyl desaturaseand kappa hydroxylase is that the kappa hydroxylase gene is expected tobe expressed more highly in tissues which have high level ofhydroxylated fatty acids than in other tissues. In contrast, all planttissues should contain mRNA for an ω6 fatty acyl desaturase sincediunsaturated fatty acids are found in the lipids of all tissues in mostor all plants.

[0135] Therefore, it was of great interest to determine whether the genecorresponding to pLesq2 was also expressed only in seeds, or is alsoexpressed in other tissues. This question was addressed by testing forhybridization of pLesq2 to RNA purified from developing seeds and fromleaves.

[0136] Total RNA was purified from developing seeds and young leaves ofL. fendleri using an Rneasy RNA extraction kit (Qiagen), according tothe manufacturer's instructions. RNA concentrations were quantified byUV spectrophotometry at λ=260 and 280 nm. In order to ensure evenloading of the gel to be used for Northern blotting, RNA concentrationswere further adjusted after recording fluorescence under UV light of RNAsamples stained with ethidium bromide and run on a test denaturing gel.

[0137] Total RNA prepared as described above from leaves and developingseeds was electrophoresed through an agarose gel containing formaldehyde(Iba et al., 1993). An equal quantity (10 μg) of RNA was loaded in bothlanes, and RNA standards (0.16-1.77 kb ladder, Gibco-BRL) were loaded ina third lane. Following electrophoresis, RNA was transferred from thegel to a nylon membrane (Hybond N+, Amersham) and fixed to the filter byexposure to UV light.

[0138] A ³²P-labelled probe was prepared from insert DNA of clone pLesq2by random priming and hybridized to the membrane overnight at 52° C.,after it had been prehybridized for 2 h. The prehybridization solutioncontained 5×SSC, 10×Denhardt's solution, 0.1 SDS, 0.1 M KPO₄ pH 6.8, 100μg/ml salmon sperm DNA. The hybridization solution had the same basiccomposition, but no SDS, and it contained 10% dextran sulfate and 30%formamide. The blot was washed once in 2×SSC, 0.5% SDS at 65° C. then in1×SSC at the same temperature.

[0139] Brief (30 min) exposure of the blot to X-ray film revealed thatthe probe pLesq2 hybridized to a single band only in the seed RNA lane(FIG. 7). The blot was re-probed with the insert from pLesq3 gene, whichgave bands of similar intensity in the seed and leaf lanes (FIG. 7).

[0140] These results show that the gene corresponding to the clonepLesq2 is highly and specifically expressed in seed of L. fendleri. Inconjunction with knowledge of the nucleotide and deduced amino acidsequence, strong seed-specific expression of the gene corresponding tothe insert in pLesq2 is a convincing indicator of the role of the enzymein synthesis of hydroxylated fatty acids in the seed oil.

[0141] Characterization of a Genomic Clone of the Gamma Hydroxylase

[0142] Genomic DNA was prepared from young leaves of L. fendleri asdescribed by Murray and Thompson (1980). A Sau3AI-partial digest genomiclibrary constructed in the vector λDashII (Stratagene, 11011 NorthTorrey Pines Road, La Jolla, Calif. 92037) was prepared by partiallydigesting 500 μg of DNA, size-selecting the DNA on a sucrose gradient(Sambrook et al., 1989), and ligating the DNA (12 kb average size) tothe BamHI-digested arms of λDashII. The entire ligation was packagedaccording to the manufacturer's conditions and plated on E. coli strainXL1-Blue MRA-P2 (Stratagene). This yielded 5×10⁵ primary recombinantclones. The library was then amplified according to the manufacturer'sconditions. A fraction of the genomic library was plated on E. coliXL1-Blue and resulting plaques (150,000) were lifted to charged nylonmembranes (Hybond N+, Amersham), according to the manufacturer'sconditions. DNA was crosslinked to the filters under UV in aStratalinker (Stratagene)

[0143] Several clones carrying genomic sequences corresponding to the L.fendleri hydroxylase were isolated by probing the membranes with theinsert from pLesq2 that was PCR-amplified with internal primers andlabelled with ³²P by random priming. The filters were prehybridized for2 hours at 65° C. in 7% SDS, 1 mM EDTA, 0.25 M Na₂HPO₄ (pH 7.2), 1% BSAand hybridized to the probe for 16 hours in the same solution. Thefilters were sequentially washed at 65° C. in solutions containing2×SSC, 1×SSC, 0.5×SSC in addition to 0.1% SDS. A 2.6 kb Xba I fragmentcontaining the complete coding sequence for the gamma-hydroxylase andapproximately 1 kb of the 5′ upstream region was subcloned into thecorresponding site of pBluescript KS to produce plasmid pLesq-Hyd andthe sequence determined completely using an automatic sequencer by thedideoxy chain termination method. Sequence data was analyzed using theprogram DNASIS (Hitachi Company).

[0144] The sequence of the insert in clone pLesq-Hyd is shown in FIGS.8A-B. The sequence entails 1855 bp of contiguous DNA sequence (SEQ IDNO:3). The clone encodes a 401 bp 5′ untranslated region (i.e.,nucleotides preceding the first ATG codon), an 1152 bp open readingframe, and a 302 bp 3′ untranslated region. The open reading frameencodes a 384 amino acid protein with a predicted molecular weight of44,370 (SEQ ID NO:4). The amino terminus lacks features of a typicalsignal peptide (von Heijne, 1985).

[0145] The exact translation-initiation methionine has not beenexperimentally determined, but on the basis of deduced amino acidsequence homology to the Castor kappa hydroxylase (noted below) isthought to be the methionine encoded by the first ATG codon atnucleotide 402.

[0146] Comparison of the pLesq-Hyd deduced amino acid sequence withsequences of membrane-bound desaturases and the castor hydroxylase(FIGS. 9A-B) indicates that pLesq-Hyd is homologous to these genes. Thisfigure shows an alignment of the L. fendleri hydroxylase (SEQ ID NO:4)with the castor hydroxylase (van de Loo et al. 1995), the Arabidopsisfad2 cDNA which encodes an endoplasmic reticulum-localized Δ12desaturase (called fad2) (Okuley et al., 1994), two soybean fad2desaturase clones, a Brassica napus fad2 clone, a Zea mays fad2 cloneand partial sequence of a R. communis fad2 clone.

[0147] The high degree of sequence homology indicates that the geneproducts are of similar function. For instance, the overall homologybetween the Lesquerella hydroxylase and the Arabidopsis fad2 desaturasewas 92.2% similarity and 84.8% identity and the two sequences differedin length by only one amino acid.

[0148] Southern Hybridization

[0149] Southern analysis was used to examine the copy number of thegenes in the L. fendleri genome corresponding to the clone pLesq-Hyd.Genomic DNA (5 μg) was digested with EcoR I, Hind III and Xba I andseparated on a 0.9% agarose gel. DNA was alkali-blotted to a chargednylon membrane (Hybond N+, Amersham), according to the manufacturer'sprotocol. The blot was prehybridized for 2 hours at 65° C. in 7% SDS, 1mM EDTA, 0.25 M Na₂HPO₄ (pH 7.2), 1% BSA and hybridized to the probe for16 hours in the same solution with pLesq-Hyd insert PCR-amplified withinternal primers and labelled with ³²P by random priming. The filterswere sequentially washed at 65° C. in solutions containing 2×SSC, 1×SSC,0.5×SSC in addition to 0.1% SDS, then exposed to X-ray film.

[0150] The probe hybridized with a single band in each digest of L.fendleri DNA (FIG. 10), indicating that the gene from which pLesq-Hydwas transcribed is present in a single copy in the L. fendleri genome.

[0151] Expression of pLesq-Hyd in Transgenic Plants

[0152] There are a wide variety of plant promoter sequences which may beused to cause tissue-specific expression of cloned genes in transgenicplants. For instance, the napin promoter and the acyl carrier proteinpromoters have previously been used in the modification of seed oilcomposition by expression of an antisense form of a desaturase (Knutsonet al. 1992). Similarly, the promoter for the β-subunit of soybeanβ-conglycinin has been shown to be highly active and to result intissue-specific expression in transgenic plants of species other thansoybean (Bray et al., 1987). Thus, although we describe the use of theL. fendleri kappa hydroxylase promoter in the examples described here,other promoters which lead to seed-specific expression may also beemployed for the production of modified seed oil composition. Suchmodifications of the invention described here will be obvious to oneskilled in the art.

[0153] Constructs for expression of L. fendleri kappa hydroxylase inplant cells are prepared as follows: A 13 kb SalI fragment containingthe pLesq-Hyg gene was ligated into the XhoI site of binary Ti plasmidvector pSLJ44026 (Jones et al., 1992) (FIG. 11) to produce plasmidpTi-Hyd and transformed into Agrobacterium tumefaciens strains GV3101 byelectroporation. Strain GV3101 (Koncz and Schell, 1986) contains adisarmed Ti plasmid. Cells for electroporation were prepared as follows.GV3101 was grown in LB medium with reduced NaCl (5 g/l). A 250 mlculture was grown to OD₆₀₀=0.6, then centrifuged at 4000 rpm (SorvallGS-A rotor) for 15 min. The supernatant was aspirated immediately fromthe loose pellet, which was gently resuspended in 500 ml ice-cold water.The cells were centrifuged as before, resuspended in 30 ml ice-coldwater, transferred to a 30 ml tube and centrifuged at 5000 rpm (SorvallSS-34 rotor) for 5 min. This was repeated three times, resuspending thecells consecutively in 30 ml ice-cold water, 30 ml ice-cold 10%glycerol, and finally in 0.75 ml ice-cold 10% glycerol. These cells werealiquoted, frozen in liquid nitrogen, and stored at −80° C.Electroporations employed a Biorad Gene pulsar instrument using cold 2mm-gap cuvettes containing 40 μl cells and 1 μl of DNA in water, at avoltage of 2.5 KV, and 200 Ohms resistance. The electroporated cellswere diluted with 1 ml SOC medium (Sambrook et al., 1989, page A2) andincubated at 28° C. for 2-4 h before plating on medium containingkanamycin (50 mg/l).

[0154]Arabidopsis thaliana can be transformed with the Agrobacteriumcells containing pTi-Hyd as described in Example 1 above. Similarly, thepresence of hydroxylated fatty acids in the transgeneic Arabidopsisplants can be demonstrated by the methods described in Example 1 above.

[0155] Constitutive Expression of the L. fendleri Hydroxylase inTransgenic Plants

[0156] A 1.5 kb EcoR I fragment from pLesq-Hyg comprising the entirecoding region of the hydroxylase was gel purified, then cloned into thecorresponding site of pBluescript KS (Stratagene). Plasmid DNA from anumber of recombinant clones was then restricted with Pst I, whichshould cut only once in the insert and once in the vector polylinkersequence. Release of a 920 bp fragment with Pst I indicated the rightorientation of the insert for further manipulations. DNA from one suchclone was further restricted with SalI, the 5′ overhangs filled-in withthe Klenow fragment of DNA polymerase I, then cut with Sac I. The insertfragment was gel purified, and cloned between the Sma I and Sac I sitesof pBI121 (Clontech) behind the Cauliflower Mosaic Virus 35S promoter.After checking that the sequence of the junction between insert andvector DNA was appropriate, plasmid DNA from a recombinant clone wasused to transform A. tumefaciens (GV3101). Kanamycin resistant colonieswere then used for in planta transformation of A. thaliana as previouslydescribed.

[0157] DNA was extracted from kanamycin resistant seedlings and used toPCR-amplify selected fragments from the hydroxylase using nestedprimers. When fragments of the expected size could be amplified,corresponding plants were grown in the greenhouse or on agar plates, andfatty acids extracted from fully expanded leaves, roots and dry seeds.GC-MS analysis was then performed as previously described tocharacterize the different fatty acid species and detect accumulation ofhydroxy fatty acids in transgenic tissues.

[0158] Expression of the Lesquerella Hydroxylase in Yeast

[0159] In order to demonstrate that the cloned L. fendleri gene encodedan oleate-12 hydroxylase, the gene was expressed in yeast cells undertranscriptional control of an inducible promoter and the yeast cellswere examined for the presence of hydroxylated fatty acids by GC-MS.

[0160] In a first step, a lambda genomic clone containing the L.fendleri hydroxylase gene was cut with EcoRI, and a resulting 1400 bpfragment containing the coding sequence of the hydroxylase gene wassubcloned in the EcoRI site of the pBluescript KS vector (Stratagene).This subclone, pLesqcod, contains the coding region of the Lesquerellahydroxylase plus some additional 3′ sequence.

[0161] In a second step, pLesqcod was cut with HindIII and XbaI, and theinsert fragment was cloned into the corresponding sites of the yeastexpression vector pYes2 (In Vitrogen; FIG. 12). This subclone, pLesqYes,contains the L. fendleri hydroxylase in the sense orientation relativeto the 3′ side of the Gal1 promoter. This promoter is inducible by theaddition of galactose to the growth medium, and is repressed uponaddition of glucose. In addition, the vector carries origins ofreplication allowing the propagation of pLesqYes in both yeast and E.coli.

[0162] Transformation of S. cerevisiae Host Strain CGY2557

[0163] Yeast strain CGY2557 (MATα, GAL⁺, ura3-52, leu2-3, trp1, ade2-1,lys2-1, his5, can1-100) was grown overnight at 28° C. in YPD liquidmedium (10 g yeast extract, 20 g bacto-peptone, 20 g dextrose perliter), and an aliquot of the culture was inoculated into 100 ml freshYPD medium and grown until the OD₆₀₀ of the culture was 1. Cells werethen collected by centrifugation and resuspended in about 200 μl ofsupernatant. 40 μl aliquots of the cell suspension were then mixed with1-2 μg DNA and electroporated in 2 mm-gap cuvettes using a Biorad GenePulser instrument set at 600 V, 200Ω, 25 μF. 160 μl YPD was added andthe cells were plated on selective medium containing glucose. Selectivemedium consisted of 6.7 g yeast nitrogen base (Difco), 0.4 g casaminoacids (Difco), 0.02 g adenine sulfate, 0.03 g L-leucine, 0.02 gL-tryptophan, 0.03 g L-lysine-HCl, 0.03 g L-histidine-HCl, 2-glucose,water to 1 liter. Plates were solidified using 1.5% Difco Bacto-agar.Transformant colonies appeared after 3 to 4 days incubation at 28° C.

[0164] Expression of the L. fendleri Hydroxylase in Yeast

[0165] Independent transformant colonies from the previous experimentwere used to inoculate 5 ml of selective medium containing either 2%glucose (gene repressed) or 2% galactose (gene induced) as the solecarbon source. Independent colonies of CGY2557 transformed with pYES2containing no insert were used as controls.

[0166] After 2 days of growth at 28° C., an aliquot of the cultures wasused to inoculate 5 ml of fresh selective medium. The new culture wasplaced at 16° C. and grown for 9 days.

[0167] Fatty Acid Analysis of Yeast Expressing the L. fendleriHydroxylase

[0168] Cells from 2.5 ml of culture were pelleted at 1800 g, and thesupernatant was aspirated as completely as possible. Pellets were thendispersed in 1 ml of 1 N methanolic HCl (Supelco, Bellafonte, Pa.).Transmethylation and derivatization of hydroxy fatty acids wereperformed as described above. After drying under nitrogen, samples wereredissolved in 50 μl chloroform before being analyzed by GC-MS. Sampleswere injected into an SP2330 fused-silica capillary column (30 m×0.25 mmID, 0.25 μm film thickness, Supelco). The temperature profile was100-160° C., 25° C./min, 160-230° C., 10° C./min, 230° C., 3 min,230-100° C., 25° C./min. Flow rate was 0.9 ml/min. Fatty acids wereanalyzed using a Hewlett-Packard 5971 series Msdetector.

[0169] Gas chromatograms of derivatized fatty acid methyl esters frominduced cultures of yeast containing pLesqYes contained a novel peakthat eluted at 7.6 min (FIG. 13). O-TMS methyl ricinoleate eluted atexactly the same position on control chromatograms. This peak was notpresent in cultures lacking pLesqYes or in cultures containing pLesqYesgrown on glucose (repressing conditions) rather than galactose (inducingconditions). Mass spectrometry of the peak (FIG. 13) revealed that thepeak has the same spectrum as O-TMS methyl ricinoleate. Thus, on thebasis of chromatographic retention time and mass spectrum, it wasconcluded that the peak corresponded to O-TMS methyl ricinoleate. Thepresence of ricinoleate in the transgenic yeast cultures confirms theidentity of the gene as a kappa hydroxylase of this invention.

Example 3 Obtaining Other Plant Fatty Acyl Hydroxylases

[0170] In a previous patent application, we described the ways in whichthe castor fah12 sequence could be used to identify other kappahydroxylases by methods such as PCR and heterologous hybridization.However, because of the high degree of sequence similarity between Δ12desaturases and kappa hydroxylases, prior art does not teach how todistinguish between the two kinds of enzymes without a functional testsuch as demonstrating activity in transgenic plants or another suitablehost (e.g., transgenic microbial or animal cells). The identification ofthe L. fendleri hydroxylase provided for the development of criteria bywhich a hydroxylase and a desaturase may be distinguished solely on thebasis of deduced amino acid sequence information.

[0171] FIGS. 9A-B show a sequence alignment of the castor and L.fendleri hydroxylase sequences with the castor hydroxylase sequence andall publicly available sequences for all plant microsomal Δ12 fatty aciddesaturases. Of the 384 amino acid residues in the castor hydroxylasesequence, more than 95% are identical to the corresponding residue in atleast one of the desaturase sequences. Therefore, none of these residuesare responsible for the catalytic differences between the hydroxylaseand the desaturases. Of the remaining 16 residues in the castorhydroxylase and 14 residues in the Lesquerella hydroxylase, all but sixrepresent instances where the hydroxylase sequence has a conservativesubstitution compared with one or more of the desaturase sequences, orthere is wide variability in the amino acid at that position in thevarious desaturases. By conservative, we mean that the following aminoacids are functionally equivalent: Ser/Thr, Ile/Leu/Val/Met, Asp/Glu.Thus, these structural differences also cannot account for the catalyticdifferences between the desaturases and hydroxylases. This leaves justsix amino acid residues where both the castor hydroxylase and theLesquerella hydroxylase differ from all of the known desaturases andwhere all of the known microsomal Δ12 desaturases have the identicalamino acid residue. These residues occur at positions 69, 111, 155, 226,304 and 331 of the alignment in FIG. 9. Therefore, these six sitesdistinguish hydroxylases from desaturases. Based on this analysis, weclaim that any enzyme with greater than 60% sequence identity to one ofthe enzymes listed in FIG. 9 can be classified as a hydroxylase if itdiffers from the sequence of the desaturases at these six positions.Because of slight differences in the number of residues in a particularprotein, the numbering may vary from protein to protein but the intentof the number system will be evident if the protein in question isaligned with the castor hydroxylase using the numbering system shownherein. Thus, in conjunction with the methods for using the Lesquerellahydroxylase gene to isolate homologous genes, the structural criteriondisclosed here teaches how to isolate and identify plant kappahydroxylase genes for the purpose of genetically modifying fatty acidcomposition as disclosed herein and in the previous application (Ser.No. 08/320,982).

[0172] In considering which of the six substitutions are solely orprimarily responsible for the difference in catalytic activity of thehydroxylases of this invention and the desaturases, we consider itlikely that the substitution of a Phe for a Tyr at position 226 may besolely responsible for this difference in catalytic activity because ofthe known participation of tyrosine radicals in enzyme catalysis. Othersubstitutions, such as the Ala for Ser at position 331 may have effectsat modulating the overall rate of the reaction. On this basis weenvision creating novel kappa hydroxylases by site directed mutagenesisof Δ12 desaturases. We also envision converting Δ15 desaturases and Δ9desaturases to hydroxylases by similar use of site-directed mutagenesis.

Example 4 Using Hydroxylases to Alter the Level of Fatty AcidUnsaturation

[0173] Evidence that kappa hydroxylases of this invention can be used toalter the level of fatty acid unsaturation was obtained from theanalysis of transgenic plants that expressed the castor hydroxylaseunder control of the Cauliflower mosaic virus promoter. The constructionof the plasmids and the production of transgenic Arabidopsis plants wasdescribed in Example 1 (above). The fatty acid composition of seedlipids from wild type and six transgenic lines (1-2/a, 1-2/b, 1-3/b, 4F,7E, 7F) is shown in Table 2. TABLE 2 Fatty acid composition of lipidsfrom Arabidopsis seeds. The asterisk (*) indicates that for some ofthese samples, the 18:3 and 20:1 peaks overlapped on the gaschromatograph and, therefore, the total amount of these two fatty acidsis reported. Fatty acid WT 1-2/a 1-2/b 1-3/b 4F 7E 7F 16:0 10.3 8.6 9.58.4 8.1 8.4 9 18:0 3.5 3.8 3.9 3.3 3.5 3.8 4.2 18:1 14.7 33 34.5 25.527.5 30.5 28.5 18:2 32.4 16.9 21 27.5 21.1 20.1 19.8 18:3 13.8 — 14.414.8 — — — 20:0 1.3 1.6 1 1.1 2.4 1.8 2 20:1 22.5 — 14.1 17.5 — — — 18:3— 31.2 — — 32.1 30.8 30.6 20:1* Ricinoleic 0 0.6 0 0.1 0.2 0.7 0.9.Densipolic 0 0.6 0 0.1 0.2 0.5 0.6 Lesquerolic 0 0.2 0 0 0.2 0.2 0.6Auricolic 0 0.1 0 0 0 0.1 0.1

[0174] The results in Table 2 show that expression of the castorhydroxylase in transgenic Arabidopsis plants caused a substantialincrease in the amount of oleic acid (18:1) in the seed lipids and anapproximately corresponding decrease in the amount of linoleic acid(18:2). The average amount of oleic acid in the six transgenic lines was29.9% versus 14.7% in the wild type.

[0175] The mechanism by which expression of the castor hydroxylase genecauses increased accumulation of oleic acid is not known. Anunderstanding of the mechanism is not required in order to exploit thisinvention for the directed alteration of plant lipid fatty acidcomposition. Furthermore, it will be recognized by one skilled in theart that many improvements of this invention may be envisioned. Ofparticular interest will be the use of other promoters which have highlevels of seed-specific expression.

[0176] Since hydroxylated fatty acids were not detected in the seedlipids of transgenic line 1-2b, it seems likely that it is not thepresence of hydroxylated fatty acids per se that causes the effect ofthe castor hydroxylase gene on desaturase activity. We speculate thatthere may be a protein-protein interaction between the hydroxylase andthe Δ12-oleate desaturase or another protein required for the overallreaction (eg., cytochrome b5) or for the regulation of desaturaseactivity. We envision that the interaction between the hydroxylase andthis other protein suppresses the activity of the desaturase. Forinstance, the quaternary structure of the membrane-bound desaturases hasnot been established. It is possible that these enzymes are active asdimers or as multimeric complexes containing more than two subunits.Thus, if dimers or multimers formed between the desaturase and thehydroxylase, the presence of the hydroxylase in the complex may disruptthe activity of the desaturase. This general hypothesis will be testeddirectly by the production of transgenic plants in which the hydroxylaseenzyme has been rendered inactive by the elimination of one or more ofthe histidine residues that have been proposed to bind iron moleculesrequired for catalysis. Several of these histidine residues have beenshown to be essential for catalysis by site directed mutagenesis(Shanklin et al., 1994). Codons encoding histidine residues in thecastor hydroxylase gene described in U.S. patent application Ser. No.08/320,982 will be changed to alanine residues as described by Shanklinet al. (1994). The modified genes will be introduced into transgenicplants of Arabidopsis and possibly other species such as tobacco by themethods described in Example 1 of this application or in Example 1 ofthe original version of this application (U.S. application Ser. No.08/320,982).

[0177] In order to examine the effect on all tissues, the strongconstitutive cauliflower mosaic virus promoter will be used to causetranscription of the modified genes. However, it will be recognized thatin order to specifically examine the effect of expression of the mutantgene on seed lipids, a seed-specific promoter such as the B. napus napinpromoter or the promoter described in Example 2, above, may be used. Anexpected outcome is that expression of the inactive hydroxylase proteinin transgenic plants will inhibit the activity of the endoplasmicreticulum-localized Δ12-desaturase. Maximum inhibition will be obtainedby expressing high levels of the mutant protein.

[0178] In a further embodiment of this invention, we envision thatmutations that inactivate other hydroxylases, such as the Lesquerellahydroxylase of this invention, will also be useful for decreasing theamount of endoplasmic reticulum-localized Δ12-desaturase activity in thesame way as the castor gene. In a further embodiment of this invention,we also envision that similar mutations of desaturase genes may be usedto inactivate endogenous desaturases. Thus, we envision that expressionof catalytically inactive fad2 gene from Arabidopsis in transgenicArabidopsis will inhibit the activity of the endogenous fad2 geneproduct.

[0179] Similarly, we envision that expression of the catalyticallyinactive forms of the Δ12-desaturase from Arabidopsis or other plants intransgenic soybean in transgenic rapeseed, Crambe, Brassica juncea,Canola, flax, sunflower, safflower, cotton, cuphea, soybean, peanut,coconut, oil palm and corn will lead to inactivation of endogenousΔ12-desaturase activity in these species. In a further embodiment ofthis invention we envision that expression of catalytically inactiveforms of other desaturases such as the Δ15-desaturases will lead toinactivation of the corresponding desaturases.

[0180] Whatever the precise basis for the inhibitory effect of thecastor hydroxylase on desaturation, because the castor hydroxylase hasvery low nucleotide sequence homology (i.e., about 67%) to theArabidopsis fad2 gene (encoding the endoplasmic reticulum-localizedΔ12-desaturase) we envision that the inhibitory effect of this gene,which we provisionally call “protein-mediated inhibition”(“protibition”), will have broad utility because it does not depend on ahigh degree of nucleotide sequence homology between the transgene andthe endogenous target gene. In particular, we envision that the castorhydroxylase may be used to inhibit the endoplasmic reticulum-localizedΔ12-desaturase activity of all higher plants. Of particular relevanceare those species used for oil production. These include but are notlimited to rapeseed, Crambe, Brassica juncea, Canola, flax, sunflower,safflower, cotton, cuphea, soybean, peanut, coconut, oil palm and corn.

[0181] Concluding Remarks

[0182] By the above examples, demonstration of is critical factors inthe production of novel hydroxylated fatty acids by expression of akappa hydroxylase gene from Castor in transgenic plants is described. Inaddition, a complete cDNA sequence of the Lesquerella fendleri kappahydroxylase is also provided. A full sequence of the castor hydroxylaseis also given with various constructs for use in host cells. Throughthis invention, one can obtain the amino acid and nucleic acid sequenceswhich encode plant fatty acyl hydroxylases from a variety of sources andfor a variety of applications. Also revealed is a novel method by whichthe level of fatty acid desaturation can be altered in a directed waythrough the use of genetically altered hydroxylase or desaturase genes.

[0183] All publications mentioned in this specification are indicativeof the level of skill of those skilled in the art to which thisinvention pertains. All publications are herein incorporated byreference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0184] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

[0185] References

[0186] Arondel, V., B. Lemieux, I. Hwang, S. Gibson, H. Goodman, C. R.Somerville. Map-based cloning of a gene controlling omega-3 fatty aciddesaturation in Arabidopsis. Science 258, 1353-1355 (1992).

[0187] Atsmon, D. (1989) Castor, in Oil Crops of the World, Robbelen,G., Downey, K. R., and Ashri, A., Eds., McGraw-Hill, New York, pp.438-447.

[0188] Bechtold, N., Ellis, J. and Pelletier, G. (1993) In PlantaAgrobacterium mediated gene transfer by infiltration of adultArabidopsis thaliana plants. C.R. Acad. Sci. Paris 316, 1194-1199.

[0189] Beltz, G. A., Jacobs, K. A., Eickbuch, T. H., Cherbas, P. T.,Kafatos, F. C. (1983) Isolation of multigene families and determinationof homologies by filter hybridization methods. Methods in Enzymology100, 266-285.

[0190] Bray, E. A., Naito, S., Pan, N. S., Anderson, E., Dube, P.,Beachy, R. N. (1987) Expression of the β-subunit of β-conglycinin inseeds of transgenic plants. Planta 172, 364-370.

[0191] Carlson, K. D., Chaudhry, A., Bagby, M. O (1990) Analysis of oiland meal from lesquerella fendleri seed. J. Am. Oil Chem. Soc. 67,438-442.

[0192] Ditta, G., Stanfield, S., Corbin, D., Helinski, D. R. (1980)Broad host range DNA cloning system for gram-negative bacteria:Construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad.Sci. USA 77, 7347-7351.

[0193] Gibson, S., Arondel, V., Iba, K., Somerville, C. R. (1994)Temperature Regulated Expression of a Gene Encoding a Chloroplastomega-3 Desaturase from Arabidopsis thaliana. Plant Physiol. 106,1615-1621.

[0194] Gould, S. J., Subramani, S., Scheffler, I. E. (1989) Use of theDNA polymerase chain reaction for homology probing. Proc. Natl. Acad.Sci. USA 86, 1934-1938.

[0195] Hirsinger, F. (1989) New oil crops, in Oil Crops of the World,Robbelen, G., Downey, K. R., and Ashri, A., Eds., McGraw-Hill, New York,pp. 518-533.

[0196] Howling, D., Morris, L. J., Gurr, M. I., James, A. T. (1972) Thespecificity of fatty acid desaturases and hydroxylases. Thedehydrogenation and hydroxylation of monoenoic acids, Biochim. Biophys.Acta 260, 10.

[0197] Huyuh, T. V., Young, R. A., Davis, R. W. (1985) Constructing andscreening cDNA libraries in λgt10 and λgt11. In DNA Cloning, Vol. 1: APractical Approach, (ed) D. M. Glover. IRL Press, Washington, D.C. pp49-77.

[0198] Iba, K., Gibson, S., Nishiuchi, T., Fuse, T., Nishimura, M.,Arondel, V., Hugly, S., and Somerville, C. (1993) A gene encoding achloroplast omega-3 fatty acid desaturase complements alterations infatty acid desaturation and chloroplast copy number of the fad7 mutantof Arabidopsis thaliana. J. Biol. Chem. 268, 24099-24105.

[0199] Jones, J. D. G., Shlumukov, L., Carland, F., English, J.,Scofield, S., Bishop, G. J., Harrison, K. (1992) Effective vectors fortransformation, expression of heterologous genes, and assayingtransposon excision in transgenic plants. Transgenic Res. 1, 285-297.

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[0201] Koncz, C., Schell, J. (1986) The promoter of T_(L)-DNA gene 5controls the tissue-specific expression of chimeric genes carried by anovel type of Agrobacterium binary vector. Mol. Gen. Genet. 204,383-396.

[0202] Matzke, M., Matzke, A. J. M. (1995) How and why do plantsinactivate homologous (Trans)genes? Plant Physiol. 107, 679-685.

[0203] Miquel, M. Browse, J. (1992) Arabidopsis mutants deficient inpolyunsaturated fatty acid synthesis. J. Biol. Chem. 267, 1502-1509.

[0204] Murray, M. G., Thompson, W. F. (1980) Rapid isolation of highmolecular weight plant DNA. Nucl. Acid Res. 8, 4321-4325.

[0205] Okuley, J., Lightner, J., Feldman, K., Yadav, N., Lark, E.,Browse, J. (1994) Arabidopsis FAD2 gene encodes the enzyme that isessential for polyunsaturated lipid. Plant Cell 6, 147-158.

[0206] Sambrook, J., Fritsch, E. F., and Maniatis, T., MolecularCloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, 1989.

[0207] Shanklin, J., Whittle, E., Fox, B. G. (1994) Eight histidineresidues are catalytically essential in a membrane-associated ironenzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylaseand xylene monoxygenase. Biochemistry 33, 12787-12794.

[0208] Smith C. R., Jr. (1985) Unusual seed oils and their fatty acids,in Fatty Acids, Pryde E. H., Ed., American Oil Chemists' Society,Champaign, Second edition, pp 29-47.

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[0212] von Heijne, G. (1985) Signal sequences. J. Mol. Biol. 184,99-105.

1 15 543 nucleotides nucleotide single linear 1 TATTGGCACC GGCGGCACCATTCCAACAAT GGATCCCTAG AAAAAGATGA AGTCTTTGTC 60 CCACCTAAGA AAGCTGCAGTCANATGGTAT GTCAAATACC TCAACAACCC TCTTGGACGC 120 ATTCTGGTGT TAACAGTTCAGTTTATCCTC GGGTGGCCTT TGTATCTAGC CTTTAATGTA 180 TCAGGTAGAC CTTATGATGGTTTCGCTTCA CATTTCTTCC CTCATGCACC TATCTTTAAG 240 GACCGTGAAC GTCTCCAGATATACATCTCA GATGCTGGTA TTCTAGCTGT CTGTTATGGT 300 CTTTACCGTT ACGCTGCTTCACAAGGATTG ACTGCTATGA TCTGCGTCTA CGGAGTACCG 360 CTTTTGATAG TGAACTTTTTCCTTGTCTTG GTCACTTTCT TGCAGCACAC TCATCCTTCA 420 TTACCTCACT ATGATTCAACCGAGTGGGAA TGGATTAGAG GAGCTTTGGT TACGGTAGAC 480 AGAGACTATG GAATCTTGAACAAGGTGTTT CACAACATAA CAGACACCCA CGTAGCACAC 540 CAC 543 544 nucleotidesnucleotide single linear 2 TATAGGCACC GGAGGCACCA TTCCAACACA GGATCCCTCGAAAGAGATGA AGTATTTGTC 60 CCAAAGCAGA AATCCGCAAT CAAGTGGTAC GGCGAATACCTCAACAACCC TCCTGGTCGC 120 ATCATGATGT TAACTGTCCA GTTCGTCCTC GGATGGCCCTTGTACTTAGC CTTCAACGTT 180 TCTGGCAGAC CCTACAATGG TTTCGCTTCC CATTTCTTCCCCAATGCTCC TATCTACAAC 240 GACCGTGAAC GCCTCCAGAT TTACATCTCT GATGCTGGTATTCTAGCCGT CTGTTATGGT 300 CTTTACCGTT ACGCTGTTGC ACAAGGACTA GCCTCAATGATCTGTCTAAA CGGAGTTCCG 360 CTTCTGATAG TTAACTTTTT CCTCGTCTTG ATCACTTACTTACAACACAC TCACCCTGCG 420 TTGCCTCACT ATGATTCATC AGAGTGGGAT TGGCTTAGAGGAGCTTTAGC TACTGTAGAC 480 AGAGACTATG GAATCTTGAA CAAGGTGTTC CATAACATCACAGACACCCA CGTCGCACAC 540 CACT 544 1855 nucleotides nucleotide singlelinear 3 ATGAAGCTTT ATAAGAAGTT AGTTTTCTCT GGTGACAGAG AAATTNTGTCAATTGGTAGT 60 GACAGTTGAA GCAACAGGAA CAACAAGGAT GGTTGGTGNT GATGCTGATGTGGTGATGTG 120 TTATTCATCA AATACTAAAT ACTACATTAC TTGTTGCTGC CTACTTCTCCTATTTCCTCC 180 GCCACCCATT TTGGACCCAC GANCCTTCCA TTTAAACCCT CTCTCGTGCTATTCACCAGA 240 AGAGAAGCCA AGAGAGAGAG AGAGAGAATG TTCTGAGGAT CATTGTCTTCTTCATCGTTA 300 TTAACGTAAG TTTTTTTTGA CCACTCATAT CTAAAATCTA GTACATGCAATAGATTAATG 360 ACTGTTCCTT CTTTTGATAT TTTCAGCTTC TTGAATTCAA GATGGGTGCTGGTGGAAGAA 420 TAATGGTTAC CCCCTCTTCC AAGAAATCAG AAACTGAAGC CCTAAAACGTGGACCATGTG 480 AGAAACCACC ATTCACTGTT AAAGATCTGA AGAAAGCAAT CCCACAGCATTGTTTCAAGC 540 GCTCTATCCC TCGTTCTTTC TCCTACCTTC TCACAGATAT CACTTTAGTTTCTTGCTTCT 600 ACTACGTTGC CACAAATTAC TTCTCTCTTC TTCCTCAGCC TCTCTCTACTTACCTAGCTT 660 GGCCTCTCTA TTGGGTATGT CAAGGCTGTG TCTTAACCGG TATCTGGGTCATTGGCCATG 720 AATGTGGTCA CCATGCATTC AGTGACTATC AATGGGTAGA TGACACTGTTGGTTTTATCT 780 TCCATTCCTT CCTTCTCGTC CCTTACTTCT CCTGGAAATA CAGTCATCGTCGTCACCATT 840 CCAACAATGG ATCTCTCGAG AAAGATGAAG TCTTTGTCCC ACCGAAGAAAGCTGCAGTCA 900 AATGGTATGT TAAATACCTC AACAACCCTC TTGGACGCAT TCTGGTGTTAACAGTTCAGT 960 TTATCCTCGG GTGGCCTTTG TATCTAGCCT TTAATGTATC AGGTAGACCTTATGATGGTT 1020 TCGCTTCACA TTTCTTCCCT CATGCACCTA TCTTTAAAGA CCGAGAACGCCTCCAGATAT 1080 ACATCTCAGA TGCTGGTATT CTAGCTGTCT GTTATGGTCT TTACCGTTACGCTGCTTCAC 1140 AAGGATTGAC TGCTATGATC TGCGTCTATG GAGTACCGCT TTTGATAGTGAACTTTTTCC 1200 TTGTCTTGGT AACTTTCTTG CAGCACACTC ATCCTTCGTT ACCTCATTATGATTCAACCG 1260 AGTGGGAATG GATTAGAGGA GCTTTGGTTA CGGTAGACAG AGACTATGGAATATTGAACA 1320 AGGTGTTCCA TAACATAACA GACACACATG TGGCTCATCA TCTCTTTGCAACTATACCGC 1380 ATTATAACGC AATGGAAGCT ACAGAGGCGA TAAAGCCAAT ACTTGGTGATTACTACCACT 1440 TCGATGGAAC ACCGTGGTAT GTGGCCATGT ATAGGGAAGC AAAGGAGTGTCTCTATGTAG 1500 AACCGGATAC GGAACGTGGG AAGAAAGGTG TCTACTATTA CAACAATAAGTTATGAGGCT 1560 GATAGGGCGA GAGAAGTGCA ATTATCAATC TTCATTTCCA TGTTTTAGGTGTCTTGTTTA 1620 AGAAGCTATG CTTTGTTTCA ATAATCTCAG AGTCCATNTA GTTGTGTTCTGGTGCATTTT 1680 GCCTAGTTAT GTGGTGTCGG AAGTTAGTGT TCAAACTGCT TCCTGCTGTGCTGCCCAGTG 1740 AAGAACAAGT TTACGTGTTT AAAATACTCG GAACGAATTG ACCACAANATATCCAAAACC 1800 GGCTATCCGA ATTCCATATC CGAAAACCGG ATATCCAAAT TTCCAGAGTACTTAG 1855 384 amino acids amino acid linear 4 Met Gly Ala Gly Gly ArgIle Met Val Thr Pro Ser Ser Lys Lys Ser 1 5 10 15 Glu Thr Glu Ala LeuLys Arg Gly Pro Cys Glu Lys Pro Pro Phe Thr 20 25 30 Val Lys Asp Leu LysLys Ala Ile Pro Gln His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser PheSer Tyr Leu Leu Thr Asp Ile Thr Leu Val Ser 50 55 60 Cys Phe Tyr Tyr ValAla Thr Asn Tyr Phe Ser Leu Leu Pro Gln Pro 65 70 75 80 Leu Ser Thr TyrLeu Ala Trp Pro Leu Tyr Trp Val Cys Gln Gly Cys 85 90 95 Val Leu Thr GlyIle Trp Val Ile Gly His Glu Cys Gly His His Ala 100 105 110 Phe Ser AspTyr Gln Trp Val Asp Asp Thr Val Gly Phe Ile Phe His 115 120 125 Ser PheLeu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Arg 130 135 140 HisHis Ser Asn Asn Gly Ser Leu Glu Lys Asp Glu Val Phe Val Pro 145 150 155160 Pro Lys Lys Ala Ala Val Lys Trp Tyr Val Lys Tyr Leu Asn Asn Pro 165170 175 Leu Gly Arg Ile Leu Val Leu Thr Val Gln Phe Ile Leu Gly Trp Pro180 185 190 Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly PheAla 195 200 205 Ser His Phe Phe Pro His Ala Pro Ile Phe Lys Asp Arg GluArg Leu 210 215 220 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val CysTyr Gly Leu 225 230 235 240 Tyr Arg Tyr Ala Ala Ser Gln Gly Leu Thr AlaMet Ile Cys Val Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Phe PheLeu Val Leu Val Thr Phe 260 265 270 Leu Gln His Thr His Pro Ser Leu ProHis Tyr Asp Ser Thr Glu Trp 275 280 285 Glu Trp Ile Arg Gly Ala Leu ValThr Val Asp Arg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His AsnIle Thr Asp Thr His Val Ala His His 305 310 315 320 Leu Phe Ala Thr IlePro His Tyr Asn Ala Met Glu Ala Thr Glu Ala 325 330 335 Ile Lys Pro IleLeu Gly Asp Tyr Tyr His Phe Asp Gly Thr Pro Trp 340 345 350 Tyr Val AlaMet Tyr Arg Glu Ala Lys Glu Cys Leu Tyr Val Glu Pro 355 360 365 Asp ThrGlu Arg Gly Lys Lys Gly Val Tyr Tyr Tyr Asn Asn Lys Leu 370 375 380 387amino acids amino acid linear 5 Met Gly Gly Gly Gly Arg Met Ser Thr ValIle Thr Ser Asn Asn Ser 1 5 10 15 Glu Lys Lys Gly Gly Ser Ser His LeuLys Arg Ala Pro His Thr Lys 20 25 30 Pro Pro Phe Thr Leu Gly Asp Leu LysArg Ala Ile Pro Pro His Cys 35 40 45 Phe Glu Arg Ser Phe Val Arg Ser PheSer Tyr Val Ala Tyr Asp Val 50 55 60 Cys Leu Ser Phe Leu Phe Tyr Ser IleAla Thr Asn Phe Phe Pro Tyr 65 70 75 80 Ile Ser Ser Pro Leu Ser Tyr ValAla Trp Leu Val Tyr Trp Leu Phe 85 90 95 Gln Gly Cys Ile Leu Thr Gly LeuTrp Val Ile Gly His Glu Cys Gly 100 105 110 His His Ala Phe Ser Glu TyrGln Leu Ala Asp Asp Ile Val Gly Leu 115 120 125 Ile Val His Ser Ala LeuLeu Val Pro Tyr Phe Ser Trp Lys Tyr Ser 130 135 140 His Arg Arg His HisSer Asn Ile Gly Ser Leu Glu Arg Asp Glu Val 145 150 155 160 Phe Val ProLys Ser Lys Ser Lys Ile Ser Trp Tyr Ser Lys Tyr Ser 165 170 175 Asn AsnPro Pro Gly Arg Val Leu Thr Leu Ala Ala Thr Leu Leu Leu 180 185 190 GlyTrp Pro Leu Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp 195 200 205Arg Phe Ala Cys His Tyr Asp Pro Tyr Gly Pro Ile Phe Ser Glu Arg 210 215220 Glu Arg Leu Gln Ile Tyr Ile Ala Asp Leu Gly Ile Phe Ala Thr Thr 225230 235 240 Phe Val Leu Tyr Gln Ala Thr Met Ala Lys Gly Leu Ala Trp ValMet 245 250 255 Arg Ile Tyr Gly Val Pro Leu Leu Ile Val Asn Cys Phe LeuVal Met 260 265 270 Ile Thr Tyr Leu Gln His Thr His Pro Ala Ile Pro ArgTyr Gly Ser 275 280 285 Ser Glu Trp Asp Trp Leu Arg Gly Ala Met Val ThrVal Asp Arg Asp 290 295 300 Tyr Gly Val Leu Asn Lys Val Phe His Asn IleAla Asp Thr His Val 305 310 315 320 Ala His His Leu Phe Ala Thr Val ProHis Tyr His Ala Met Glu Ala 325 330 335 Thr Lys Ala Ile Lys Pro Ile MetGly Glu Tyr Tyr Arg Tyr Asp Gly 340 345 350 Thr Pro Phe Tyr Lys Ala LeuTrp Arg Glu Ala Lys Glu Cys Leu Phe 355 360 365 Val Glu Pro Asp Glu GlyAla Pro Thr Gln Gly Val Phe Trp Tyr Arg 370 375 380 Asn Lys Tyr 385 383amino acids amino acid linear 6 Met Gly Ala Gly Gly Arg Met Pro Val ProThr Ser Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Thr Thr Lys Arg Val ProCys Glu Lys Pro Pro Phe Ser 20 25 30 Val Gly Asp Leu Lys Lys Ala Ile ProPro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu IleSer Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Asn TyrPhe Ser Leu Leu Pro Gln Pro 65 70 75 80 Leu Ser Tyr Leu Ala Trp Pro LeuTyr Trp Ala Cys Gln Gly Cys Val 85 90 95 Leu Thr Gly Ile Trp Val Ile AlaHis Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu AspAsp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro TyrPhe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr GlySer Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Gln Lys SerAla Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly ArgIle Met Met Leu Thr Val Gln Phe Val Leu Gly Trp Pro Leu 180 185 190 TyrLeu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Phe Ala Cys 195 200 205His Phe Phe Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu Gln 210 215220 Ile Tyr Leu Ser Asp Ala Gly Ile Leu Ala Val Cys Phe Gly Leu Tyr 225230 235 240 Arg Tyr Ala Ala Ala Gln Gly Met Ala Ser Met Ile Cys Leu TyrGly 245 250 255 Val Pro Leu Leu Ile Val Asn Ala Phe Leu Val Leu Ile ThrTyr Leu 260 265 270 Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser SerGlu Trp Asp 275 280 285 Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg AspTyr Gly Ile Leu 290 295 300 Asn Lys Val Phe His Asn Ile Thr Asp Thr HisVal Ala His His Leu 305 310 315 320 Phe Ser Thr Met Pro His Tyr Asn AlaMet Glu Ala Thr Lys Ala Ile 325 330 335 Lys Pro Ile Leu Gly Asp Tyr TyrGln Phe Asp Gly Thr Pro Trp Tyr 340 345 350 Val Ala Met Tyr Arg Glu AlaLys Glu Cys Ile Tyr Val Glu Pro Asp 355 360 365 Arg Glu Gly Asp Lys LysGly Val Tyr Trp Tyr Asn Asn Lys Leu 370 375 380 384 amino acids aminoacid linear 7 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser LysLys Ser 1 5 10 15 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys Glu Thr ProPro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys PheLys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser His Leu Ile Trp Asp Ile IleIle Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu LeuPro Asn Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala CysGln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys GlyHis Ala Ala Phe 100 115 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val GlyLeu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp LysTyr Ser His Arg Arg His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu ArgAsp Glu Val Phe Val Pro Arg 145 150 155 160 Arg Ser Gln Thr Ser Ser GlyThr Ala Ser Thr Ser Thr Thr Phe Gly 165 170 175 Arg Thr Val Met Leu ThrVal Gln Phe Thr Leu Gly Trp Pro Leu Tyr 180 185 190 Leu Ala Phe Asn ValSer Gly Arg Pro Tyr Asp Gly Gly Phe Ala Cys 195 200 205 His Phe His ProAsn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu Gln 210 215 220 Ile Tyr IleSer Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu Leu 225 230 235 240 ProTyr Ala Ala Val Gln Gly Val Ala Ser Met Val Cys Phe Leu Arg 245 250 255Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr Leu 260 265270 Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp Asp 275280 285 Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile Leu290 295 300 Asn Gln Gly Phe His Asn Ile Thr Asp Thr His Glu Ala His HisLeu 305 310 315 320 Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala ThrLys Ala Ile 325 330 335 Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp GlyThr Pro Val Val 340 345 350 Lys Ala Met Trp Arg Glu Ala Lys Glu Cys IleTyr Val Glu Pro Asp 355 360 365 Arg Gln Gly Glu Lys Lys Gly Val Phe TrpTyr Asn Asn Lys Leu Xaa 370 375 380 309 amino acids amino acid linear 8Ser Leu Leu Thr Ser Phe Ser Tyr Val Val Tyr Asp Leu Ser Phe Ala 1 5 1015 Phe Ile Phe Tyr Ile Ala Thr Thr Tyr Phe His Leu Leu Pro Gln Pro 20 2530 Phe Ser Leu Ile Ala Trp Pro Ile Tyr Trp Val Leu Gln Gly Cys Leu 35 4045 Leu Thr Arg Val Cys Gly His His Ala Phe Ser Lys Tyr Gln Trp Val 50 5560 Asp Asp Val Val Gly Leu Thr Leu His Ser Thr Leu Leu Val Pro Tyr 65 7075 80 Phe Ser Trp Lys Ile Ser His Arg Arg His His Ser Asn Thr Gly Ser 8590 95 Leu Asp Arg Asp Glu Arg Val Lys Val Ala Trp Phe Ser Lys Tyr Leu100 105 110 Asn Asn Pro Leu Gly Arg Ala Val Ser Leu Leu Val Thr Leu ThrIle 115 120 125 Gly Trp Pro Met Tyr Leu Ala Phe Asn Val Ser Gly Arg ProTyr Asp 130 135 140 Ser Phe Ala Ser His Tyr His Pro Tyr Arg Val Arg LeuLeu Ile Tyr 145 150 155 160 Val Ser Asp Val Ala Leu Phe Ser Val Thr TyrSer Leu Tyr Arg Val 165 170 175 Ala Thr Leu Lys Gly Leu Val Trp Leu LeuCys Val Tyr Gly Val Pro 180 185 190 Leu Leu Ile Val Asn Gly Phe Leu ValThr Ile Thr Tyr Leu Arg Val 195 200 205 His Tyr Asp Ser Ser Glu Trp AspTrp Leu Lys Gly Ala Leu Ala Thr 210 215 220 Met Asp Arg Asp Tyr Gly IleLeu Asn Lys Val Phe His His Ile Thr 225 230 235 240 Asp Thr His Val AlaHis His Leu Phe Ser Thr Met Pro His Tyr His 245 250 255 Leu Arg Val LysPro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Asp Thr 260 265 270 Pro Phe TyrLys Ala Leu Trp Arg Glu Ala Arg Glu Cys Leu Tyr Val 275 280 285 Glu ProAsp Glu Gly Thr Ser Glu Lys Gly Val Tyr Trp Tyr Arg Asn 290 295 300 LysTyr Leu Arg Val 305 302 amino acids amino acid linear 9 Phe Ser Tyr ValVal Tyr Asp Leu Thr Ile Ala Phe Cys Leu Tyr Tyr 1 5 10 15 Val Ala ThrHis Tyr Phe His Leu Leu Pro Gly Pro Leu Ser Phe Arg 20 25 30 Gly Met AlaIle Tyr Trp Ala Val Gln Gly Cys Ile Leu Thr Gly Val 35 40 45 Trp Val ValAla Phe Ser Asp Tyr Gln Leu Leu Asp Asp Ile Val Gly 50 55 60 Leu Ile LeuHis Ser Ala Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr 65 70 75 80 Ser HisArg Arg His His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu 85 90 95 Val PheVal Pro Lys Val Ser Lys Tyr Leu Asn Asn Pro Pro Gly Arg 100 105 110 ValLeu Thr Leu Ala Val Thr Leu Thr Leu Gly Trp Pro Leu Tyr Leu 115 120 125Ala Leu Asn Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala Cys His Tyr 130 135140 Asp Pro Tyr Gly Pro Ile Tyr Ser Val Ile Ser Asp Ala Gly Val Leu 145150 155 160 Ala Val Val Tyr Gly Leu Phe Arg Leu Ala Met Ala Lys Gly LeuAla 165 170 175 Trp Val Val Cys Val Tyr Gly Val Pro Leu Leu Val Val AsnGly Phe 180 185 190 Leu Val Leu Ile Thr Phe Leu Gln His Thr His Val SerGlu Trp Asp 195 200 205 Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg AspTyr Gly Ile Leu 210 215 220 Asn Lys Val Phe His Asn Ile Thr Asp Thr HisVal Ala His His Leu 225 230 235 240 Phe Ser Thr Met Pro His Tyr His AlaMet Glu Ala Thr Val Glu Tyr 245 250 255 Tyr Arg Phe Asp Glu Thr Pro PheVal Lys Ala Met Trp Arg Glu Ala 260 265 270 Arg Glu Cys Ile Tyr Val GluPro Asp Gln Ser Thr Glu Ser Lys Gly 275 280 285 Val Phe Trp Tyr Asn AsnLys Leu Ala Met Glu Ala Thr Val 290 295 300 372 amino acids amino acidlinear 10 Met Gly Ala Gly Gly Arg Met Thr Glu Lys Glu Arg Glu Lys GlnGlu 1 5 10 15 Gln Leu Ala Arg Ala Thr Gly Gly Ala Ala Met Gln Arg SerPro Val 20 25 30 Glu Lys Pro Pro Phe Thr Leu Gly Gln Ile Lys Lys Ala IlePro Pro 35 40 45 His Cys Phe Glu Arg Ser Val Leu Lys Ser Phe Ser Tyr ValVal His 50 55 60 Asp Leu Val Ile Ala Ala Ala Leu Leu Tyr Phe Ala Leu AlaIle Ile 65 70 75 80 Pro Ala Leu Pro Ser Pro Leu Arg Tyr Ala Ala Trp ProLeu Tyr Trp 85 90 95 Ile Ala Gln Gly Ala Phe Ser Asp Tyr Ser Leu Leu AspAsp Val Val 100 105 110 Gly Leu Val Leu His Ser Ser Leu Met Val Pro TyrPhe Ser Trp Lys 115 120 125 Tyr Ser His Arg Arg His His Ser Asn Thr GlySer Leu Glu Arg Asp 130 135 140 Glu Val Phe Val Pro Lys Lys Lys Glu AlaLeu Pro Trp Tyr Thr Pro 145 150 155 160 Tyr Val Tyr Asn Asn Pro Val GlyArg Val Val His Ile Val Val Gln 165 170 175 Leu Thr Leu Gly Trp Pro LeuTyr Leu Ala Thr Asn Ala Ser Gly Arg 180 185 190 Pro Tyr Pro Arg Phe AlaCys His Phe Asp Pro Tyr Gly Pro Ile Tyr 195 200 205 Asn Asp Arg Glu ArgAla Gln Ile Phe Val Ser Asp Ala Gly Val Val 210 215 220 Ala Val Ala PheGly Leu Tyr Lys Leu Ala Ala Ala Phe Gly Val Trp 225 230 235 240 Trp ValVal Arg Val Tyr Ala Val Pro Leu Leu Ile Val Asn Ala Trp 245 250 255 LeuVal Leu Ile Thr Tyr Leu Gln His Thr His Pro Ser Leu Pro His 260 265 270Tyr Asp Ser Ser Glu Trp Asp Trp Leu Arg Gly Ala Leu Ala Thr Met 275 280285 Asp Arg Asp Tyr Gly Ile Leu Asn Arg Val Phe His Asn Ile Thr Asp 290295 300 Thr His Val Ala His His Leu Phe Ser Thr Met Pro His Tyr His Ala305 310 315 320 Met Glu Ala Thr Lys Ala Ile Arg Pro Ile Leu Gly Asp TyrTyr His 325 330 335 Phe Asp Pro Thr Pro Val Ala Lys Ala Thr Trp Arg GluAla Gly Glu 340 245 350 Cys Ile Tyr Val Glu Pro Glu Asp Arg Lys Gly ValPhe Trp Tyr Asn 355 360 365 Lys Lys Phe Xaa 370 224 amino acids aminoacid linear 11 Trp Val Met Ala His Asp Cys Gly His His Ala Phe Ser AspTyr Gln 1 5 10 15 Leu Leu Asp Asp Val Val Gly Leu Ile Leu His Ser CysLeu Leu Val 20 25 30 Pro Tyr Phe Ser Trp Lys His Ser His Arg Arg His HisSer Asn Thr 35 40 45 Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys LysLys Ser Ser 50 55 60 Ile Arg Trp Tyr Ser Lys Tyr Leu Asn Asn Pro Pro GlyArg Ile Met 65 70 75 80 Thr Ile Ala Val Thr Leu Ser Leu Gly Trp Pro LeuTyr Leu Ala Phe 85 90 95 Asn Val Ser Gly Arg Pro Tyr Asp Arg Phe Ala CysHis Tyr Asp Pro 100 105 110 Tyr Gly Pro Ile Tyr Asn Asp Arg Glu Arg IleGlu Ile Phe Ile Ser 115 120 125 Asp Ala Gly Val Leu Ala Val Thr Phe GlyLeu Tyr Gln Leu Ala Ile 130 135 140 Ala Lys Gly Leu Ala Trp Val Val CysVal Tyr Gly Val Pro Leu Leu 145 150 155 160 Val Val Asn Ser Phe Leu ValLeu Ile Thr Phe Leu Gln His Thr His 165 170 175 Pro Ala Leu Pro His TyrAsp Ser Ser Glu Trp Asp Trp Leu Arg Gly 180 185 190 Ala Leu Ala Thr ValAsp Arg Asp Tyr Gly Ile Leu Asn Lys Val Phe 195 200 205 His Asn Ile ThrAsp Thr Gln Val Ala His His Leu Phe Thr Met Pro 210 215 220 20nucleotides nucleotide single linear 12 GCTCTTTTGT GCGCTCATTC 20 20nucleotides nucleotide single linear 13 CGGTACCAGA AAACGCCTTG 20 20nucleotides nucleotide single linear 14 TAYWSNCAYM GNMGNCAYCA 20 21nucleotides nucleotide single linear 15 RTGRTGNGCN ACRTGNGTRT C 21

What is claimed is:
 1. An isolated nucleic acid fragment comprising anucleic acid sequence encoding a fatty acid hydroxylase with an aminoacid identity of 60% or greater to the polypeptide encoded by SEQ IDNO:4.
 2. The isolated nucleic acid fragment of claim 1, wherein theamino acid identity is 90% or greater to the polypeptide encoded by SEQID NO:4.
 3. The isolated nucleic acid fragment of claim 1, wherein theamino acid identity is 100% of the polypeptide encoded by SEQ ID NO:4.4. An isolated nucleic acid fragment having a nucleic acid identity of90% or greater of a nucleotide sequence of SEQ ID NO:1, 2, or
 3. 5. Anisolated nucleic acid having a nucleotide sequence of SEQ ID NO:1, SEQID NO:2 or SEQ ID NO:3.
 6. The isolated nucleic acid fragment of claim1, wherein said fragment is isolated from an oil-producing plantspecies.
 7. A chimeric gene capable of causing altered levels ofricinoleic acid in a transformed plant cell, said chimeric genecomprising a nucleic acid fragment of claim 1, said fragment operablylinked to suitable regulatory sequences.
 8. A chimeric gene capable ofcausing altered levels of lesquerolic acid in a transformed plant cell,said chimeric gene comprising a nucleic acid fragment of claim 1, saidfragment operably linked to suitable regulatory sequences.
 9. A chimericgene capable of causing altered levels of fatty acids in a transformedplant cell, said chimeric gene comprising a nucleic acid fragment ofclaim 1, said fragment operably linked to suitable regulatory sequences.10. A chimeric gene capable of causing altered levels of fatty acids ina transformed plant cell, said chimeric gene comprising a nucleic acidfragment of claim 2, said fragment operably linked to suitableregulatory sequences.
 11. A chimeric gene capable of causing alteredlevels of fatty acids in a transformed plant cell, said chimeric genecomprising a nucleic acid fragment of claim 4, said fragment operablylinked to suitable regulatory sequences.
 12. Plants containing thechimeric gene of any one of claims 7, 8, 9, 10 or
 11. 13. Oil obtainedfrom seeds of the plants of claim
 12. 14. The isolated nucleic acidfragment of claim 1, wherein said fragment is obtainable from Ricinuscommunis (L.) (Castor).
 15. The isolated nucleic acid fragment of claim1, wherein said fragment is obtainable from Lesquerella fendleri.
 16. Amethod of producing seed oil containing altered levels of hydroxylatedfatty acids comprising: (a) transforming a plant cell of anoil-producing species with a chimeric gene containing an isolatednucleic acid of claim 1; (b) growing fertile plants from the transformedplant cells of step (a); (c) screening progeny seeds from the fertileplants of step (b) for the desired levels of hydroxylated fatty acids;and (d) processing the progeny seed of step (c) to obtain seed oilcontaining altered levels of hydroxylated fatty acids.
 17. The method ofclaim 16, wherein said plant is selected from the group consisting ofrapeseed, Crambe, Brassica juncea, Canola, flax, sunflower, safflower,cotton, cuphea, soybean, peanut, coconut, oil palm and corn.
 18. Amethod of producing seed oil containing altered levels of hydroxylatedfatty acids comprising: (a) transforming a plant cell of anoil-producing species with a chimeric gene containing the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; (b) growing fertileplants from the transformed plant cells of step (a); (c) screeningprogeny seeds from the fertile plants of step (b) for the desired levelsof hydroxylated fatty acids; and (d) processing the progeny seed of step(c) to obtain seed oil containing altered levels of unsaturated fattyacids.
 19. The method of claim 18, wherein said plant is selected fromthe group consisting of rapeseed, Crambe, Brassica juncea, Canola, flax,sunflower, safflower, cotton, cuphea, soybean, peanut, coconut, oil palmand corn.
 20. A triglyceride oil from a plant selected from the groupconsisting of rapeseed, Crambe, Brassica juncea, Canola, flax,sunflower, cotton, cuphea, soybean, peanut, coconut, oil palm and corn,wherein the fatty acid composition of the oil has been modified tocontain hydroxylated fatty acids by a method comprising growing a plantcell having integrated in its genome a DNA construct containing a planthydroxylase encoding sequence of claim 1, under conditions which willpermit the transcription and translation of said plant hydroxylase inthe plant cells.
 21. A method to isolate nucleic acid fragments encodingfatty acid hydroxylases comprising: (a) comparing SEQ ID NO:4 and otherfatty acid hydroxylase sequences and fatty acid desaturases; (b)identifying conserved sequences of 4 or more amino acids obtained instep (a); (c) designing degenerate oligomers based on the conservedsequences identified in step (b); (d) using the degenerate oligomers ofstep (c) to isolate sequences encoding fatty acid hydroxylases bysequence dependent protocols; (e) obtaining the deduced amino acidsequence of the encoded gene product from the nucleotide sequence of thegene and; (f) distinguishing hydroxylase genes from desaturase genes byanalyzing amino acid sequence differences between fatty acid desaturasesand fatty acid hydroxylases.
 22. A method of producing seed oilcontaining altered levels of unsaturated fatty acids comprising: (a)transforming a plant cell of an oil-producing species with a chimericgene containing an isolated nucleic acid comprising a nucleic acidsequence encoding a fatty acid hydroxylase with an amino acid identityof 60% or greater to the polypeptide encoded by SEQ ID NO:4; (b) growingfertile plants from the transformed plant cells of step (a); (c)screening progeny seeds from the fertile plants of step (b) for thedesired levels of unsaturated fatty acids; and (d) processing theprogeny seed of step (c) to obtain seed oil containing altered levels ofunsaturated fatty acids.
 23. A chimeric gene capable of causing alteredlevels of oleic acid in a transformed plant cell, said chimeric genecomprising a nucleic acid sequence encoding a fatty acid hydroxylasewith an amino acid identity of 60% or greater to the polypeptide encodedby SEQ ID NO:4 and operably linked to regulatory sequences.
 24. Achimeric gene capable of causing altered levels of oleic acid in atransformed plant cell, said chimeric gene comprising a nucleic acidsequence encoding a fatty acid hydroxylase with an amino acid identityof 60% or greater to the polypeptide encoded by SEQ ID NO:4 in whichdirected changes have been made which lead to the replacement of one ormore essential histidine residues in the corresponding gene product,said chimeric gene operably linked to regulatory sequences.
 25. Themethod of claims 22, 23 or 24, wherein said plant is selected from thegroup consisting of rapeseed, Crambe, Brassica juncea, Canola, flax,sunflower, safflower, cotton, cuphea, soybean, peanut, coconut, oil palmand corn.